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ASAI Kiyofumi

    Executives　Vice Chairperson/President
Last Updated :2024/04/26

Researcher Information

URL

J-Global ID

Research Areas

  • Life sciences / Fetal medicine/Pediatrics
  • Life sciences / Neuroscience - general

Academic & Professional Experience

  • 1998 - 2001  Nagoya City University
  • 1998 - 2001  Nagoya
  • 2001  - 名古屋市立大学医学部
  • 2001  - Nagoya
  • 1989 - 1998  Nagoya City University
  • 1989 - 1998  Nagoya

Education

  •        - 1989  Nagoya City University  医学研究科  小児科学
  •        - 1989  Nagoya City University  Graduate School, Division of Medicine
  •        - 1984  Nagoya City University  医学部  医学科
  •        - 1984  Nagoya City University  Faculty of Medicine

Association Memberships

  • 日本神経科学学会   The International Society for Developmental Neuroscience   The Society for Study of Inborn Errors of Matebolism   日本先天代謝異常学会   日本小児科学会   日本神経化学会   日本癌学会   日本生化学会   

Published Papers

Books etc

  • Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenation
    Brain Res Mol Brain Res 2001
  • Expression of glia maturation factor during retinal development in the rat
    Brain Res Mol Brain Res 2001
  • Recurrent subthreshold electrical activities of rat neocortical neurons progress during long-term culture
    Neurosci Lett 2001
  • Isolation and characterization of a new immortal rat astrocyte with a high expression of NGF mRNA
    Neurosci Res 2001
  • Acid stimulates E-cadherin surface expression on gastric epithelial cells to stabilize barrier functions via influx of calcium
    Eur J Gastroenterol Hepatol 2001
  • Interleukin-1beta induces the expression of lipocortin 1 mRNA in cultured rat cortical astrocytes
    Neurosci Res 2001
  • Biosynthetic response of cultured articular chondrocytes to mechanical vibration
    Res Exp Med(Berl) 2001
  • Effects of mechanical vibration on DNA and proteoglycan synthesis in cultured artcular chondrocytes.
    Mod Pheumatol 2001
  • Alpha-fetoprotein producing gastric cancer lacks transcription factor ATBF1
    Oncogene 2001
  • A metabotropic glutamate receptor antagonist, alpha-methyl-4-carboxyphenylglycine, attenuates immediate early gene mRNA expression following traumatic injury in cultured rat cortical glial cells
    Neurosci Lett 2001
  • Cyclic ADP-ribose as a potential second messenger for neuronal Ca(2+)signaling
    J Neurochem 2001
  • Regulation of rat hippocampal neural cadherin in the kainic acid induced seizures
    Neurosci Lett 2001
  • Human neuroblastomas with unfavorable biologies express high levels of brain-derived neurotrophic factor mRNA and a variety of its variants
    Cancer Lett 2001
  • Regulation of aquaporin-4 expression in astrocytes
    Brain Res Mol Brain Res 2001
  • Calcium oxalate crystal attachment to cultured rat kidney epithelial cell
    nrk-52e, Urol Int 2001
  • Differential regulation of aquaporin expression in astrocytes
    Brain Res Mol Brain Res 2001
  • Synovial inflammation and hyperplasia induced by gliostatin/platelet-derived endothelial cell growth factor in rabbit knees
    Rheumatol Int 2000
  • Cloning of a rat glia maturation factor-gamma(rGMFG)cDNA and expression of its mRNA and protein in rat organs
    J Biochem(Tokyo) 2000
  • AT motif binding factor 1-A(ATBF1-A)negatively regulates transcription of the aminopeptidase N gene in the crypt-villus axis of small intestine
    Biochem Biophys Res Commun 2000
  • p27Kip1 expression by contact inhibition as a prognostic index of human glioma
    J Neurochem 2000
  • Abnormal glycosylation of IgG as a clinical parameter in patients with rheumatoid arthritis : Its constitutional analysis by HPLC
    J Clin Biocehm Nutr 1998
  • Gliostatin/platelet-derived endothelial cell growth factor as a clinical marker of rheumatoid arthritis and its regulation in fibroblast-like synoviocytes
    Br J Rheumatol 1997
  • Glucocorticoid induced the expression of mRNA and the secretion of lipocortin 1 in rat astrocytoma cells
    Brain Res 1997
  • Evidence for participation of gliostatin/platelet-derived endothelial cell growth factor in gastric ulcer healing
    Life Sci 1997
  • Specific detection of kappa light chain in uric acid stones
    Life Sci 1997
  • Tissue distribution of human gliostatin/platelet-derived endothelial cell growth factor(PD-ECGF)and its drug-induced expression
    Biochim Biophys Acta 1996
  • Astrocytic contributions to blood-brain barrier(BBB)formation by endothelial cells : a possible use of aortic endothelial cell for in vitro BBB model, Neurochem Int
    Neurochem Int 1996
  • Heat shock-mediated cell cycle arrest is accompanied by induction of p21 CKI
    Biochem Biophys Res Commun 1996
  • Aberrant production of gliostatin/platelet-derived endothelial cell growth factor in rheumatoid synovium
    Arthritis Rheum 1994
  • Growth-promoting action of adenosine-containing dinucleotide on neuroblastoma cells : detection of adenosine-cytidine dinucleotide (ApCp) in neurofibroma (NF1) extracts.
    J Neurochem 1993

MISC

Research Grants & Projects

  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2020/04 -2023/03 
    Author : 永谷 祐子; 浅井 清文; 川口 洋平; 野崎 正浩
     
    本研究の目的は、既存の治療薬に対して不応性のRA患者へグリオスタチンを標的とした新たな治療薬を開発することである。これまでの臨床研究にてTNF阻害 薬、IL-6阻害剤、Janus kinase (JAK) 阻害剤治療中患者の血清中グリオスタチン濃度の推移を観察すると、これら既存の治療薬 に対して寛解導入できた症例で は血清中グリオスタチン濃度が、低下したが、不応性の患者では血清中グリオスタチン濃度は高濃度で推移することがわかっている。またRAにおいては血清のグ リオスタチンは滑膜にて産生される。RA関節液中のグリオスタチン濃度は変形性膝関節症患者の数十倍から数百倍におよびこのグリオスタチンを制御するには抗体療法ではなくシグナル伝達阻害が効率的であると考え、グリオスタチンを標的とした治療法の開発を試みる。 申請者らはグリオスタチンプロモーターを組み込んだvectorを作製し、 線維芽細胞様滑膜細胞 (FLSs) に導入しグリオスタチン プロモーター活性を測定した。 グリオスタチンプロモーターにはSp1結合部位が7か所あり、Sp1阻害剤であるmithramycin (MIT) にてプロモーター活性が抑制されることを確認した。またグリオスタチンプロモーターにはSTAT1の結合部位であるISRE、 GASがあり、同部位を標的にした活性抑制可能な因子の探索を試みた。グリオスタチンはinterferon gamma刺激にても誘導されたことからISRE、 GASは治療標的となりうることが示唆された。今回はさらにグリオスタチンの発現がSTATのリン酸化がどのように関連しているのかを明らかにする。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/04 -2023/03 
    Author : 岡本 秀貴; 川口 洋平; 永谷 祐子; 浅井 清文
     
    4週令Wistarラットの手指および足趾から爪母を採取して37℃，10分のcollagenase処理を行った。その爪母を37℃、CO2:10%で処理して爪母間様細胞を単離培養することに成功した。3継代目の爪母間様細胞をmitomycin C処理してfeeder layerとした。そこに4週令Wistarラットの手指および足趾から採取した爪母を蒔いて培養した。こうして爪母上皮細胞を単離培養することに成功した。爪母上皮細胞を抗ハードケラチン抗体（Keratin 17/19 (D4G2) XP Rabbit mAb）を用いて免疫染色を行ったところ抗ハードケラチン抗体陽性で爪母上皮細胞であることが証明された。 これらの細胞を用いてgroup 1は3系代目の爪母間様細胞をコラーゲンゲルに混ぜて培養して、そのコラーゲンゲルの上面に爪母上皮細胞を蒔いた群。group 2はコラーゲンゲル内と上面に爪母間様細胞のみを蒔いた群、group 3はコラーゲンゲルのみの群とした。group 4はポリカプロラクトン（PCL）で作製されたスキャフォードに爪母間様細胞を蒔いた群、group 5はPCLで作製されたスキャフォードに爪母上皮細胞を蒔いた群、group 6はPCLのみの群とした。これらを5日間培養した後でWistarラット背部皮下に移植した。3週間後に移植組織を摘出して抗ハードケラチン抗体（Keratin 17/19 (D4G2) XP Rabbit mAb）を用いて免疫染色を行った。 group 1、2ではケラチンを産生し抗ハードケラチン抗体陽性であった。しかし、産生されたハードケラチンは層構造を成すものの、各層間には間隙があって剥がれそうな構造を呈していた。group 4、5では何らかの産生物はあったが抗ハードケラチン抗体陰性であった。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2018/04 -2022/03 
    Author : Yoshihito Fujita
     
    We developed that acquaporin knockdown cell lines and over-expression cell lines and we also demonstrated that bupivacaine induces neural death with calcium channel type T of bupivacaine. We confirmed that with the specific incubator that could make accurate low O2 condition, our cell lines could make grow in 1% O2 condition and that means that hypoxic preconditioning could be established. Moreover, we combined this 1% O2 hypoxic condition and low glucose level and we established neural ischemic models. We could proceed nest steps for our experiments.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2017/04 -2021/03 
    Author : Asai Kiyofumi
     
    To detect the cellular polarity of astrocyte, we employed aquaporin-4 (AQP4) expression as a marker of polarity. To detect the intracellular distribution of AQP4, pHluorin and mKate double-labeled AQP4 expression vector was constructed. pHluorin was introduced to the extracellular loop of AQP4 and mKate was fused to C-terminal. This vector was transfected to C6 astrocytoma and ACT57 immortalized rat astrocyte. Both green signal (pHluorin) and red signal (mKate) were detected 48 to 96 hrs after transfection. When the extracellular pH was changed from pH7.4 to pH6.0, the green signal was disappeared. This result might suggest AQP4 fusion protein was expressed on the plasma membrane. Next, the co-culture system of bovine brain endothelial cell line tBBEC-117 and ACT57 was tested. Clear cellular polarity was not detected at present.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2017/04 -2020/03 
    Author : Nagaya Yuko
     
    The treatment of rheumatoid arthritis (RA) is in the era of remission. However, some patients have sustained local synovitis despite systemic inflammation has subsided. We have reported that gliostatin is closely involved in the development of RA. Intra-articular injection of high molecular weight hyaluronic acid (HMW-HA) is widely used as a local treatment for osteoarthritis (OA) and RA, however the mechanism of action on synoviocytes is still unclear . In this study, we used cultured synoviocytes to clarify the mechanism by which HMW-HA controls angiogenic factors such as gliostatin and vascular endothelial cell growth factor, and to effectively and safely prevent synovitis and joint destruction. The significance of topical treatment with HMW-HA was found.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2016/04 -2019/03 
    Author : Kobayashi Masaki
     
    We found for the first time that gliostatin is closely involved in the pathogenesis of rheumatoid arthritis. In this study, the goal is to apply the signal cascade control of gliostatin to the treatment of rheumatoid arthritis. We have searched for signal cascade regulators of gliostatin. There are seven Sp1 binding sites in the gliostatin promoter region, and the expression of gliostatin was inhibited by inhibiting Sp1. The knock-on effects were examined using mithramycin, which is an Sp1 transcription factor inhibitor. We also clarified that matrix metalloproteinases-1, 3, 9, 13 that induce cartilage destruction were suppressed by mithramycin.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2016/04 -2019/03 
    Author : Aoyama Mineyoshi
     
    Periventricular leukomalacia (PVL) is a white matter brain injury. PVL is specific for the premature infants with the greatest risk of the cerebral palsy. Erythropoietin (EPO), a hematopoietic hormonal cytokine induced in response to hypoxia, has neuroprotective effects. In our present study, we investigated whether EPO could attenuate the microglia activation, developing the neuroprotection. These data indicate that EPO treatment attenuates microglial activation including morphological change, phagocytosis, and the production of inflammatory cytokines in vitro and in vivo. Further investigation of EPO modulation of the microglial activation may contribute to the development of novel neuroprotective therapies.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2018/03 
    Author : SOBUE Kazuya; ISHII Fumiyasu; YAMAMOTO Naoki
     
    Recently, patients with dementia such as Alzheimer type dementia (AD) are increasing. Anesthesia against AD has a possibility of deteriorating cognitive function after surgery. The pathological future of AD is senile plaque due to aggregation of amyloid β (Aβ) and neurofibrillary tangle (microtubule aggregation, Tau abnormal phosphorylation). In this study, propofol, barbital and midazolam showed little influence on accumulation of Aβ in cultured neurons and mouse brain, whereas ketamine and haloperidol could be promoted. In addition, as a preventive measure, SKF 10047 which is a σ 1 receptor agonist and epigallocatechin were found to promote Aβ degradation. This research provides a clue to the development of safe anesthesia and preventive measures against AD.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2018/03 
    Author : Fujita Yoshihito
     
    For making aquaporin (AQP) 4 knock down, we used Lentiviral RNAi system of Invitrogen corporation. Finally, we obtained AQP 4 knockdown model with pLenti4/BLOCK-IT/Expression Construct and plasmid of pLP1、pLP2、pLP/VSVG、and pENTER-gus. We confirmed that AQP4 knockdown that was stronger than our earlier experiment. We constructed hAQP4-M23 and hAQP4-M1 vector and transfected into C6 cells. We confirmed the overexpression of hAQP4-M1 and hAQP4-M23 in C6 cells. Moreover, we constructed permanent cell line of hAQP4-M23-overexpressed-cells of C6.In hypoxic condition, we tried to decide apoptosis or necrosis with flow cytometry. For optimal oxygen concentration, we experimented cell culture of astrocyte and SH-SY5Y in hyperoxia. We confirmed cell damage of hyperoxia in this condition. We try to continue neuron protection of various condition and drugs using RNAi technique.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2015/04 -2018/03 
    Author : Kakita Hiroki
     
    Hypothermia has been reported to be only effective therapy for neonatal hypoxic ischemic encephalopathy (HIE) . We performed the examination of microenviroment improvement of glial function control with hypothermia. We found hypothermia (32-34℃) decreased the expression of iNOS and inflammatory cytokine in cultured glia under hypoxic condition. Furthermore, erythropoietin having neuroprotective effect made clear that overexpression lasted by a low temperature state. We found that these protein expression adjustment by hypothermia decreased brain damage. This study might contribute to the development of an etiology of HIE which is still a poor prognosis and the cure.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2014/04 -2018/03 
    Author : Imaizumi Yuji; Higuchi Tsunehiko; Asai Kiyofumi; Hirono Syuichi
     
    The present study revealed that in non-excitable cells such as chondrocytes andendothelial cells, Ca2+-activated K+ (KCa) channels and store-operated Ca2+ (SOC) channels play significant roles in the positive feedback mechanism for the regulation of intracellular Ca2+ concentration ([Ca2+]i). This [Ca2+]i elevation forms cellular responses to various types of stimulations in physiological conditions or gets involved in pathological process. In chondrocytes, it is demonstrated that ion channels, such as large-conductance Ca2+-activated K+ (BK) channels, Ca2+-release-activated Ca2+ (CRAC) channels, ClC-3 and ClC-7, are involved in progression of osteoarthritis. In brain capillary endothelial cells, hypoxic stress induces the up-regulation of Kir2.1, augment of positive-feedback mechanisms and promotion of cell proliferation. These events may play a role in disruption of blood-brain-barrier after cerebral hypoxia.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2014/04 -2017/03 
    Author : NAGAYA Yuko; KAWAGUCHI Yohei; OGURI Yusuke; TATEMATSU Naoe
     
    Treatment of rheumatoid arthritis (RA) has advanced dramatically in the last decade, and it has become possible to aim for remission through strong treatment intervention such as biological and targeted anti-rheumatic drugs. However, there are drug-resistant patients who do not respond to any drug treatment. We have reported for the first time that gliostatin is closely involved in the pathogenesis of RA. In this study, we clarified that this gliostatin can be therapeutically targeted. The mechanism of joint destruction by gliostatin and its suppression mechanism were analyzed by molecular biological method using fibroblast-like synoviocytes derived from patients with RA
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2013/04 -2017/03 
    Author : Iizuka Narushi; HAYANO Junichiro; AKASHI Keiko; ASAI Kiyofumi; SUZUI Masumi; SUZUI Masumi
     
    In both East Japan great earthquake disaster stricken area and non-stricken area, recognition degree of prescribed medicine was investigated. The recognition rate of the drug name prescribed for chronic diseases was very low (< 20%). The possession rate of the medicine notebook was extensively higher rate at a stricken area than a non-stricken area. This was the successful result of instruction and education to the patients at the hospitals and the drugstores. The investigation at the stricken area was carried out with undergraduate students related to medical care. Educational effects on disaster risk reduction to students were one big result. Meanwhile, the cooperation between the healthcare workers among different organizations could be anti-disaster measures. The ICLS course was found to be effective on personnel interchange where the healthcare workers of many medical institutions participated in. This indicated that the course may greatly contribute to disaster risk reduction.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2013/04 -2016/03 
    Author : Aoyama Mineyoshi; ASAI KIYOFUMI
     
    Recently, it has been demonstrated that erythropoietin (EPO), a hematopoietic hormone, has a neuroprotective effect and EPO receptor (EPOR) is expressed in the central nervous system (CNS). In our previous study, brain immune cells microglia had higher expressions of EPOR than astrocytes and neurons. In vitro , we assessed the effect of EPO using microglial cell line BV-2 activated by lipopolysaccharide(LPS). LPS increased the gene expressions of cytokines in BV-2, but these increases were significantly suppressed by EPO. Next, LPS treatment increased the phagocytosis in BV-2 compared with untreated BV-2, but this increase was significantly suppressed by EPO. In vivo, LPS induced microglia to be activated types, but EPO alleviated the active morphological change. These data indicated that LPS made microglia activated and cytotoxic, but EPO-EPOR signal might attenuate LPS-induced microglial cytotoxic activation.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2015/03 
    Author : TAKESHI Sugiura; SOBUE Kazuya; ASAI Kiyofumi
     
    The Aim of this study is to identify the specific biomarker for neuropathic pain, as for diagnosis of neuropathic pain and evaluation of therapeutic efficacy. We confirmed the hyperalgesia-like behavior in the pain animal model. The use of cerebrospinal fluid as a sample to measure the biomarker is more challenging than spinal, peripheral nerve and blood. We could have not identified the appropriate protein to analyze for biomarker of neuropathic pain due to technical difficulties for adjustment of materials.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2015/03 
    Author : ARIMA Hajime; ASAI Kiyofumi; SOBUE Kazuya
     
    Hypercapnia has been reported to have a protective effect for ventilator induced lung injury, however the detail is unknown. To demonstrate a mechanism of protective effect of the hypercapnia for the ventilator induced lung injury, mice model of ventilator induced lung injury was developed. Large amount of ventilatory volume increased lung injury in a time dependent manner. Hypercapnia significantly decreased the ventilator induced lung injury. Cytokines such as IL-1b, IL-6, MIP-1a and TNFa from bronchoalveolar lavage solution were measured. Interleukin-6 was significantly increased by the artificial ventilation and significantly decreased by hypercapnia. JAK-STAT signal pathway, which is downstream pathway from IL-6, was associated with mechanism of protective effect of the hypercapnia to the ventilator induced lung injury. These results raise hopes developing a new drug for the ventilator induced lung injury.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2015/03 
    Author : SOBUE Kazuya; SUGIURA Takeshi; ASAI Kiyofumi
     
    The use of anesthetics for the Alzheimer's type dementia (AD) patients, there is a possibility that exacerbate the pathology. The purpose of this study was to clarify the effects of anesthetic drugs on the pathology of AD, it is to establish a secure anesthesia methods. As the pathogenesis of AD, it has been found that GM1 ganglioside is to promote the polymerization of amyloid β (Aβ). Propofol, thiopental, midazolam will become apparent to suppress the expression of GM1, it is likely to be used safely. Ketamine reduces the activity of neprilysin are enzymes that degrade Aβ, may promote Aβ accumulation. As a result of the above it is useful in the establishment of safe anesthesia for AD patients.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2015/03 
    Author : FUJITA Yoshihito; SOBUE Kazuya; ASAI Kiyofumi
     
    Using vector expressing shRNA, we constructed lentivirus expressing sh RNAiof aquaporin(AQP) 4. We confirmed AQP4 knockdown that was stronger than our earlier study. We constructed hAQP4-M23 and hAQP4-M1 vector and transfected into C6 cells. We confirmed the overexpression of hAQP4-M1 and hAQP4-M23 in C6 cells. Moreover, we constructed permanent cell line of hAQP4-M23-overexpressed-cells of C6.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2012/04 -2015/03 
    Author : OTSUKA Takanobu; NAGAYA Yuko; ASAI Kiyofumi
     
    Aquaporins (AQPs) are usually present at the plasma membrane, where they regulate the influx and outflow of water and small molecules. We screened for the expression of 12 AQP mRNAs (AQP1-AQP12) in the synovial tissues of patients with osteoarthritis (OA) and rheumatoid arthritis (RA), and examined the patterns of expression in those with and without hydrarthrosis. AQP1, AQP3 and AQP9 were expressed in synovial tissues from patients with OA and RA. Fibroblast-like synoviocytes (FLSs) from patients with RA and OA were cultured and stimulated with TNF-α. AQP9 mRNA expression was strongly induced by TNF-α in FLSs, whereas expression of AQP1 and AQP3 mRNAs was not influenced by TNF-α. Treatment of hyaluronic acid did not affected AQP9 mRNA expression induced by TNF-α. Although the functions of AQP1, AQP3 and AQP9 in synovial tissues remain to be elucidated, AQP9 might play a role in hydrarthrosis and inflammatory synovitis.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011/11 -2014/03 
    Author : IMAIZUMI Yuji; OHYA Susumu; YAMAMURA Hisao; HIGUCHI Tsunehiko; ASAI Kiyofumi; HIRONO Syuichi
     
    The present study revealed that, in non-excitable cells such as endothelial cells, lymphocytes, chondrocytes and airway cilliated cell, Ca2+-activated K+ (KCa) channels and store-operated Ca2+ (SOC) channels play significant roles in the positive feedback mechanism for the regulation of intracellular Ca2+ concentration ([Ca2+]i). This [Ca2+]i elevation elicites cellular responses to various types of stimula under physiological conditions or is involved in pathological processes. In brain capillary endothelial cells and chondrocytes, Ca2+-release activated Ca2+ channel is a major component of SOC channels and regulates cell proliferation. In T lymphocytes isolated from inflammatory disease model mice, the change in expression level of intermediate-conductance KCa channel is related to pathological processes. In ciliated cells, ATP-sensitive K+ channel contribute to the positive feedback mechanism for [Ca2+]i, and facilitate ciliary movement and consequently promote airway clearance.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011 -2013 
    Author : NAGAYA Yuko; OTSUKA Takanobu; ASAI Kiyofumi
     
    Gliostatin/thymidine phosphorylase (GLS/TP) has angiogenic and arthritogenic activities, and aberrant GLS production has been observed in the active synovial membranes of rheumatoid arthritis (RA) patients. The human GLS gene promoter contains at least seven consensus binding sites for the DNA binding protein Sp1. Here we examined whether Sp1 is necessary for GLS production in RA.Mithramycin is an aureolic-acid anti-neoplastic antibiotic used for treating cancer-related hypercalcaemia, leukemia and testicular cancer, which prevents Sp1 binding to its cognate site in DNA by modifying CG sequences. We also studied the effects of the Sp1 inhibitor mithramycin on GLS production in RA fibroblast-like synoviocytes.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2011 -2013 
    Author : ASAI Kiyofumi; AOYAMA Mineyoshi; KAKITA Hiroki
     
    To understand the pathophysiological mechanism of acute encephalopathy, and analyze the aggregative action of diclofenac sodium (DCF) on acute encephalopathy, we stimulated primary cultured rat astrocytes and microglia with IL-1beta;, TNF-alpha and IFN-gamma. The expression of iNOS and AQP4 and the production of NO were upregulated in astrocytes and the expression of iNOS and the activities of phagocytosis were increased in microglia. The addition of DCF highly enhanced the expression of iNOS and AQP4, and the activities of phagocytosis. This enhancement may explain the significantly increased mortality rates in acute encephalopathy patients treated with DCF.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2010 -2012 
    Author : SOU Mine; ASAI Kiyofumi; SOBUE Kazuya; ARIMA Hajime; HIRATE Hiroyuki
     
    Astrocytic swelling is one of the mechanisms of brain edema. We found that AQP4 enhanced expression which rose by ischemia-reoxygenation relates to astrocytic swelling. Participation of AQP4 was suggested to the cerebral edema by brain ischemia in an animal model. When astrocyte was medicated with the inhibitory drug(SN-50) of NF-kB, or the inhibitory drug(SB203580)of p38 MAPK and ischemia-reoxygenation was caused, the tendency for the rapid revelation rise of AQP4 to be controlled was acquired. Although the further examination was required, a possibility that regulation of cerebral edema could be performed by this research was suggested.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2010 -2012 
    Author : HIRATE Hiroyuki; SUGIURA Takeshi; ASAI Kiyofumi; SOBUE Kazuya
     
    It is still difficult to know the relationship between occurrence of the failure to the central nerve system, and brain edema where the water channel aquaporin 4 is believed to play an important role, which is accompanied by severe infection. The study for this subject is still in progress.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2010 -2012 
    Author : AOYAMA Mineyoshi; ASAI Kiyofumi
     
    We have made human neural stem cell from human iPS cell and investigated whether the induction of exogenous oncogene N-myc could transform the cell or not. We would prepare human or mouse neural stem cells at various step of the development. And N-myc genes were induced in the cells. The effect on cell proliferation was observed. These studies may reflect the cellular transformation from the normal cells to the cancer cells to clarify the mechanism of the carcinogenesis.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2010 -2012 
    Author : KUBOTA Eiji; KATAOKA Hiromi; ASAI Kiyofumi; AOYAMA Mineyoshi; SUZUKI Syugo
     
    Despite of recent advances in therapies for cancers, it is not sufficient to improve the outcome of advanced gastric cancer patients. Therefore, it is indispensable to develop novel target molecules for gastric cancer treatment. We have previously demonstrated that ES cell-expreesed Ras, ERas is expressed in gastric cancer, and is involved in gastric cancer invasiveness and metastasis. Our data also indicated the significance of ERas as a predict biomarker for gastric cancer patients. In this study, we showed the mechanisms by which ERas enhances the metastatic ability, and a utility of ERas as a target of gastric cancer treatment.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2009 -2011 
    Author : SOBUE Kazuya; ASAI Kiyofumi; SUGIURA Takeshi
     
    The membrane pore proteins, aquaporins(AQPs), facilitate the osmotically driven passage of water and, in some instances, small solutes. AQP4 plays important roles in maintaining brain homeostasis and formation of brain edema. However, little is known about function of AQP9 in brain edema. The expression of AQP9 is regulated by the p38 MAPK pathway. Hypoxia evoked a marked decrease in the expression levels of AQP9 and in astrocytes in vitro subsequent reoxygenation elicited the restoration of the expression of AQP9 to their basal levels. These results suggest that AQP9 may be one of the candidates for inducing the intracranial edema in the CNS after ischemia injury.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2009 -2011 
    Author : FUJITA Yoshihito; ASAI Kiyofumi; SOBUE Kazuya
     
    We constructed hAQP4-M23 and hAQP4-M1 vector and transfected into C6 cells. We confirmed the overexpression of hAQP4-M1 and hAQP4-M23 in C6 cells. Moreover, we constructed permanent cell line of hAQP4-M23-overexpressed-cells of C6.We constructed vector of knockdown for AQP4, however, we could not obtain efficacy of knockdown for AQP4 using our constructed vectors. We tried a new method using lentiviral vectors
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2008 -2010 
    Author : ASAI Kiyofumi; AOYAMA Mineyoshi
     
    We have tried to establish immortalized mouse brain endothelial cell lines and rat astrocytes cell line for the co-culture system of both cells. Primary cultured mouse brain endothelial cells were transfected with SV40 T-antigen expressing vector and selected by zeocin and blasticidin. Primary cultured rat astrocytes were transfected with same vectors and selected by some procedures. Three colonies from endothelial cells and 10 colonies from astrocytes were obtained, but in all of the immortalized cell lines, expression of SV40 T was not controlled by tetracycline. Thus Tet-repressor expressing vector was re-transfected to the cell lines and finally 3 astrocyte cell lines were obtained in which expression of SV40 T was controlled by tetracycline. Now we are investigating whether the expression of GFAP changes or not by on/off of SV40 T, and trying to establish co-culture system of endothelial cell lines and astrocyte cell lines.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2008 -2010 
    Author : IMAIZUMI Yuji; OHYA Susumu; YAMAMURA Hisao; ASAI Kiyofumi; HIGUCHI Tunehiko
     
    The present study revealed that functional expression of Ca^<2+>-activated K+ (BK, IK, SK) channels in non-excitable cells, such as chondrocytes, vascular endothelial cells and T-lymphocytes, substantially contributes to sustained increase in intracellular Ca^<2+> concentration in response to various types of stimuli. We proved that Ca^<2+>-activated K+ channels play the central roles in the positive feedback mechanism for the regulation of intracellular Ca^<2+> concentration via the membrane hyperpolarization, which increases the driving force of store-operated Ca^<2+> influx through non-selective cation channels.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2008 -2009 
    Author : 久保田 英嗣; 片岡 洋望; 浅井 清文
     
    われわれが独自に作製したヒトERas抗体を用いた免疫組織染色を胃癌142症例に行い,ERas発現を臨床病理学的に検討した.ERas発現と組織型の間には有意な相関は認めなかったが,リンパ節転移(p<0.05)および肝転移(p<0.001)と有意な正の相関を認めた.分子生物学的検討からは,ERas発現により胃癌細胞の転移・浸潤能が増強することが示された.またERas発現による胃癌細胞の形態変化,FibronectinやVimentinなどの間葉系マーカーの発現増強,E-cadherinなどの上皮マーカーの発現抑制などから,ERasは腫瘍転移の重要なメカニズムの一つとされている上皮間葉移行を誘導することが示唆された.以上の結果から胃癌においてERasは,上皮間葉移行を誘導することにより転移能を獲得していると考えられた.なおsiRNAを用いた検討では,ERasのkockdownにより間葉上皮移行が誘導される結果が得られた. ヌードマウス(BALB/c Slc-nulnu)を用いた腫瘍皮下移植モデルの検討では,ERas高発現胃癌細胞株で腫瘍形成能および増殖能が有意に増強されていた.また免疫不全マウス(Nod-skid mouse)を用いた転移モデルの検討から,ERas高発現胃癌細胞株では有意に転移能が増強されており,臨床病理学的検討および分子生物学的検討の結果を支持するものであった. 以上の結果から,胃癌においてERasは,浸潤・転移を誘導する重要な因子の一つであり,胃癌の予後診断マーカー,胃癌治療の標的因子となりうると考えられた.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2007 -2009 
    Author : AOYAMA Mineyoshi; ASAI Kiyofumi
     
    We had a plan to isolate putative cancer stem cells from mouse neuroblastoma cell lines. And also we would isolate mouse neural stem cells from mouse embryonal and adult brain. Following the isolation of both stem cells, we would find the similarity and the difference between neuroblastoma cancer stem cells and neural stem cells. We have isolated mouse neural stem cells. However, we have failed to isolate neuroblastoma cancer stem cells using the sorting method by the specific cell surface marker or the neurosphere method. We suggest that after frequent passages, cancer cell lines could lose the cancer stem cells, which can be found in human primary tumor.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2007 -2008 
    Author : OTSUKA Takanobu; NAGAYA Yuko; ASAI Kiyofumi
     
    整形外科疾患では種々の病態により関節水腫の発症をみる.日常臨床においては, 関節穿刺, 副腎皮質ホルモンの関節内投与, ヒアルロン酸の関節内投与, 関節鏡視下滑膜切除術などの対症療法がとられることが多く, しばしば再発する.関節水腫をきたす個々の疾患たとえば関節リウマチ(RA), 変形性関節症(OA)などの疾患解明は著しい進展しているものの, 関節水腫そのもの発生機序の分子機構の解明にはあまり進展はない.そこで本研究では水チャネルのアクアポリン(APQ)を介しての関節水腫発症の分子機構を解明し, 効果的治療法の開発をめざした RA、OA にて人工関節置換術を施行した際に得られた滑膜組織についてAPQ の発現を検討した. 滑膜組織のmRNA を抽出し、real time PCRで解析したところAPQ-1, 3, 9 の発現を認めた。APQ-1,3ついてはRA, OAでは発現の差は認めなかったが、APQ-9についてはRA において高発現の傾向にあった。OA 滑膜では、術前水腫ありの滑膜では、水腫なしの滑膜よりAPQ-9 の発現が高かった。滑膜組織を抗 APQ-1,3,9抗体にて免疫組織染色したところ、滑膜表層細胞、血管内皮細胞にAPQ-1,3,9が発現していることがわかった 滑膜から培養した線維芽細胞様滑膜培養細胞を炎症性サイトカインであるTumornecrosis factor (TNF)-αにて刺激するとAQP-9 が誘導され、炎症による水腫発症にAQP-9 が関与していることが示唆された
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2006 -2008 
    Author : FUJITA Yoshihito; ASAI Kiyofumi; SOBUE Kazuya
     
    今後の研究の基礎となるアクアポリン(AQP)Knockdown細胞株の確立を重点に行った。Knockdownが確認されているconstructに加えオリジナルのものを作成した。vectorには、invitrogen社製のBlock-IT Pol II miR RNAi expression vector、pcDNA 6.2-GW/miRを使用した。そのプラスミドを大腸菌にtransformationして、大量培養し、Quiagen社のキットを用いて、目的となるconstructを含むプラスミドを大量に、無菌的に精製した。RNAiの効果を確認するが効果が弱く、原因の追求のためGFPをvectorに挿入した。そのGFPつきのvectorの挿入で、視覚的におおざっぱにいって20%ぐらいのtranfection効率があることを確認した。ただRNAi効果をえる効率は、まだ10%程度にとどまった。transfection効率をあげるためレンチウイルスを使用したvectorの作製を進めると同時に、現在作成してあるGFPつきvectorを用い。実際の低温における脳浮腫効果の解明を始めている。また、同時に疾患の重傷度の指標として注目している乳酸値について、乳酸負荷でのアストロサイトにおける水チャンネルの発現の変化についても解析し、論文発表した。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2006 -2008 
    Author : SOBUE Kazuya; ASAI Kiyofumi; SUGIURA Takeshi
     
    水チャネルであるアクアポリン(AQP)は、赤血球において水を特異的に通過させるチャネルである。脳にはAQP4が豊富に発現しており、AQP4が脳浮腫に深く関与している可能性が示唆されている。本研究により、アストロサイトにおいてRIL(reversion-inducedLIM)がAQP4と結合し、AQP4の細胞内分布や機能を調節している可能性がある。新規脳浮腫治療法への応用が期待できる。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2006 -2007 
    Author : NAGAYA Yuko; OTSUKA Takanobu; ASAI Kiyofumi
     
    Gliostatin/thymidine phosphorylase (GLS/TP) is known to have angiogenic and arthritogenic activities. We previously demonstrated, for the first time, significantly higher concentrations of GLS/TP in the sera and synovial fluids of patients with rheumatoid arthritis (RA) compared to those with osteoarthritis or normal controls. In cultured RA fibroblast-like synoviocytes (FLSs), GLS expression was found to be up-regulated by inflammatory cytokines, such as IL-i and tumour-necrosis factor (TNF)α. The purpose of this study was to elucidate whether GLS/TP is involved in the regulation of the angiogenic cytokine vascular endothelial growth factor (VEGF) in RA. Fibroblast-like synoviocytes (FLSs) from patients with RA were cultured and stimulated with recombinant human GLS (rHuGLS) and interleukin (IL)-1β. Immunohistochemistry showed that GLS/TP and VEGF were detectable in the synovial lining cells from RA patients. GLS/TP and VEGF were even more weakly detected in synovial specimens from osteoarthritis patients than these from RA patients. In cultured FLSs, both VEGF mRNA and protein levels were markedly increased by rHuIL-1β treatment. rHuGLS increased VEGF mRNA expression in a dose-dependent manner. We detected high concentrations of VEGF165 protein in culture supernatants from FLSs treated with rHuGLS (300 ng/ml), which were comparable to GLS levels found in synovial fluid of RA patients. These findings indicate that GLS/TP and VEGF have synergistic effects on angiogenesis in rheumatoid synovitis, and that GLS/TP has a role in regulating VEGF
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2006 -2007 
    Author : 浅井 清文; 三浦 裕; 青山 峰芳
     
    申請者らは,アストロサイトが様々な脳内部位において異なる機能を有することを想定し,その相違を分子レベルで証明することを目的に解析を行ってきた。昨年までに,凍結ラット脳切片を迅速免疫組織染色法によって抗GFAP抗体にて染色し,laser microdissection(LMD)法を用いて各脳内部位からGFAP陽性細胞のみを採取し,遺伝子発現の違いをDNA microarray(Agilent社製)を用いて比較した。その中で大脳皮質と線条体において有意に発現の違いを認めた遺伝子,12個を見いだした。そこで,今年度は詳細な観察を行うため,大脳皮質で発現が高かった水の輸送に関与するアクアポリン4(AQP4)の発現について注目し解析を行った。空間的,時間的多様性を調べるために年齢別,部位別に凍結ラット脳切片を用いた免疫組織染色を行った。また,年齢別,部位別のアストロサイト培養細胞を用いた免疫細胞染色を行い,細胞培養系においても脳内部位特異性が保持されるか検討した。その結果,少なくとも大脳皮質と線条体においてアストロサイトにおけるAQP4の染色性が異なることがわかり,それは部位特異的に均一な細胞集団であるというより,部位特異的にヘテロな細胞集団の構成が異なることが考えられた。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2005 -2007 
    Author : 竹内 昭憲; 伊藤 弘晃; 祖父江 和哉; 浅井 清文; 竹内 昭憲
     
    頭部外傷や脳血管障害による、血液脳関門の破綻や神経細胞障害に対する有効な治療法はいまだ確立されていない。本研究は、内皮細胞前駆細胞(endothelial progenitor cell: EPC)を使用し、破綻した脳血管の再生を試みようとするものである。さらに、内皮細胞として定着した前駆細胞に神経栄養因子遺伝子を導入しておくことにより、損傷した神経細胞への遺伝子治療を目指したい。これらの研究により、損傷により破綻した血管(血液脳関門)と神経細胞障害を同時に治療できる可能性がある。EPCのマーカーであるCD34を指標として、抗CD34抗体を吸着させたマグネットビーズを使用し、抗原抗体反応を利用して単離を行った。同方法により、マウスの血液からEPCを分離したところ、CD34陽性であることを確認した。単離の精度は向上し、比較的純度の高い細胞を得られるようになった。さらに、単離したEPCを脳内環境におくことで、脳毛細血管内皮細胞へ分化するかどうかを調査した。アストロサイト培養上清を収集し、その上清をEPC培養液中に添加し、Flk-1/KDRやTie-2/Tek、GLUT-1などの脳毛細血管内皮細胞のマーカーが発現の確認を試みた。しかしながら、十分な分化を認めなかったため、アストロサイトの培養上清の濃縮を行った。濃縮された上清をEPC培養液中に添加したが、やはり十分な分化が得られなかったため、現在更なる濃縮を試みている。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2005 -2006 
    Author : ASAI Kiyofumi; MIURA Yutaka
     
    1, Analysis of expression of glia maturation factor beta after cryogenic injury We investigated the expression of GMFB during 56 days after cryogenic brain injury, using immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and enzyme immunoassay. Immunohistochemical analysis demonstrated that the GFAP-positive astrocytes around the lesion expressed GMFB protein, peaking 14 days after injury. Weak astrocytic expression of GMFB immunoreactivity was seen in sham-operated animal brains. Cryogenic injury (CI) induced GMFB mRNA in the lesioned side after 7 days with a maximum at 14 days. Western blotting revealed the induction of GMFB protein starting 1 day after injury, and continuing until 14 days after injury. In the enzyme immunoassay, GMFB protein concentration peaked 14 days after injury in extracts from the injured side of the brain, whereas in serum it peaked 1 day after injury. These data indicate that the expression of GMFB increased in the astrocytes around the lesioned area after cortical cryogenic brain injury. 2, Analysis of promoter activity of the human glia maturation factor-gamma gene We determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5'-flanking region of the first exon has no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5'RACE (rapid amplification of cDNA 5' ends) identified multiple transcription initiation sites within the region-84 to-70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells. We have also determined the organization of the mouse GMFG gene and cloned the 5'-flanking region of the first exon. The promoter activity was analyzed using luciferase assay.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2005 -2006 
    Author : KATAOKA Hiromi; JOH Takashi; ASAI Kiyofumi; OHARA Hirotaka
     
    Background and aims: ERas, a novel member of the ras family, was found to be expressed in ES cells only. ERas is constitutively active without any mutations and plays a crucial role when transplanted in the transformation of ES cells to teratomas and the expression level of ERas gradually decreases when ES cells are induced to differentiate. We investigated the expression and the roles of ERas, in human gastric cancer cells. Results: 1. ERas was expressed in human gastric cancer and almost all gastric cancer cell lines. 2. Overexpression of ERas did not induce the phosphorylation of MEK. However, overexpression of ERas induced phosphorylation of Akt. 3. The ERas transfectants were significantly more resistant to CPT-11 than the parent lines (survival rate: control 23.0%, the ERas transfectants 67.4%, p <0.001), but not to 5-Fluorouracil, Cisplatin, and Paclitaxel. Overexpression of ERas significantly reduced CPT-11-induced apoptosis (p <0.01). 4. Administration of Rapamycin significantly induced cytotoxicity to the ERas transfectants (survival rate: control 93.9%, ERas transfectant 68.5%, p <0.01). 5. ERas was expressed in about 30% in human gastric cancer. Overexpression of ERas did not induce the phosphorylation of MEK but induced phosphorylation of Akt. 6. Microarray analysis revealed that ERas induced down-regulation of E-cadherin and up-regulation of ABCG2 which is capable of extruding CTPT-11 from cells. Gene set analyses of microarray indicated that ERas induced activation of NF-κB. E-cadherin inhibition was also confirmed at protein levels by westernblot and ERas significantly promoted colony-formation in soft agar, with about a 4-fold number of colonies as compared with control. Conclusion: ERas was expressed in human gastric cancer and gastric cell lines, and plays crucial roles for anti-apoptosis via phosphorylation of Akt. ERas may be associated with proliferation and metastasis of gastric cancer through inhibition of E-cadherin and activation of NFκB.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2005 -2006 
    Author : YAMADA Kazuo; KATANO Hiroyuki; AIHARA Noritaka; MASE Mitsuhito; ASAI Kiyofumi
     
    We have studied mRNA and protein expression of myelencephalon-specific protease (MSP) in the brain ischemia and brain injury model in rats, because MSP is oligodendrocyte-specific serine protease and MSP expression is relating to axonal regeneration. As a result, we have detected MSP mRNA and protein expression in the oligodendrocytes locating at peri-ischemic area 3 to 7 days after cerebral ischemia. Question then arises whether MSP expression is caused by direct ischemic injury to the peri-ischemic oligodendrocytes or indirect effect of edema fluid formation on oligodendrocytes. To answer this question, we developed cold injury model in which edema developed around lesion but ischemic injury to the oligodendrocyte was not occurring. We detected in the cold injury model that MSP expression occurred on the 5^ day after cold injury. With this result we concluded that edema fluid extending to the surrounding lesion might activate oligodendrocytes and express MSP mRNA and protein expression. We did double immunostaining of MSP and oligodendrocyte-specific marker CNPase, and identified that both markers were co-localizing in the same oligodendrocyte. The results suggest that edema fluid activates oligodendrocytes and act for regeneration of axons. We also did Western blotting of the MSP and identified that MSP proteins has bands at 19kDa (authentic MSP protein) and 37, 40 and 50kDa's. The 37kDa bands increased its volume at 6 hours and 5-7days after cold injury. Those two peaks of reactive production suggested two mechanism of oligodendrocytes activation, initial direct stimulation through neuronal network and secondary indirect stimulation through edema fluid extension. That similar result was also identified in the subarachnoid hemorrhage model and intracerebral hemorrhage model.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2004 -2005 
    Author : NAGAYA Yuko; OTSUKA Takanobu; ASAI Kiyohumi
     
    Neovascularization, proliferation of synovial cells, and mononuclear cell influx and activation are characteristic events observed in synovial joints in the pathohistology of rheumatoid arthritis (RA). We have previously measured the concentration of the angiogenic factor, gliostatin (GLS), in sera and synovial fluids of RA patients, and demonstrated for the first time an enormously high concentration in RA synovial fluids as well as in RA sera. Furthermore our previous study demonstrated that GLS acts as a cytokine to augment its own synthesis in fibroblast-like synoviocytes (FLSs) obtained from patients with RA through an autocrine mechanism. It should be noted that GLS additionally caused induction and extracellular secretion of matrix metalloproteinase (MMP)-1 and MMP-3 triggering cartilage degeneration. Recently we reported that intraarticular injection of rHuGLS to rabbit knees induced RA-like synovitis. The purpose of this study was to elucidate whether GLS/TP is involved in the regulation of the angiogenic cytokine vascular endothelial growth factor (VEGF) in rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs) from patients with RA were cultured and stimulated with recombinant human GLS (rHuGLS) and interleukin (IL)-1β. Immunohistochemistry showed that GLS/TP and VEGF were detectable in the synovial lining cells. In cultured FLSs, both VEGF mRNA and protein levels were markedly increased by rHuIL-1βtreatment. rHuGLS increased VEGF mRNA expression in a dose-dependent manner. We detected high concentrations of VEGF165 protein in culture supernatants from FLSs treated with rHuGLS (300ng/ml), which were comparable to GLS levels found in synovial fluid of RA patients. These findings indicate that GLS/TP and VEGF have synergistic effects on angiogenesis in rheumatoid synovitis, and that GLS/TP has a role in regulating VEGF.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2004 -2005 
    Author : KATANO Hiroyuki; MASE Mitsuhito; UMEMURA Atsushi; AIHARA Noritaka; YAMADA Kazuo; ASAI Kiyofumi
     
    MSP(myelencephalic-specific protease) is often found in human brain and spinal white matter, especially in oligodendrocytes, microglias and neurons. Trypsin-like activity leads to the possibility of function related to regeneration of the basement membrane and extracellular matrix, growth of oligodendrocyte, hydolysis of myelin, neural growth and synaptic plasticity. We confirmed expression of MSP mRNA in rat brain using middle cerebral artery occlusion model. In situ hybridization (ISH) of MSP mRNA demonstrated a higher level in the corpus callosum and around the ischemic area from 12h to 14days after MCA reperfusion, with the peak of expression coming 3days after reperfusion in both regions. Immunohistochemically, the expression of protein was found 1day autoradiography, immunostaining and double immunohistochemical labeling revealed the expression of MSP to be located mainly in the oligodendrocytes. Analysis of MSP mRNA after cryogenic injury by ISH revealed a higher level of expression around the cryogenic area than on the contralateral side at 2-7 days after injury, with peak expression occurring 7days. Immunohistochemical analysis demonstrated expression of MSP protein at 1day after injury, in the area around the lesion. Double immunohistochemical labeling revealed that MSP was expressed mainly in oligodendrocytes. These results suggest that expression of MSP may be related to the turnover of myelin-associated proteins and extracellular matrix proteins after cold injury. We performed quantitative evaluation of white matter damage in diffuse axonal injury (DAI) with silver impregnation method using modified Marmarou's model, which revealed the number of dark axons at the cerebral peduncle increased during the experimental period. There was a significant difference between the number of dark axons 180 days after the injury and 1, 3, 7, 30, or 60 days after the injury. Since this model showed 20% motality rate, we decided to employ DAI model using fluid percussion injury with higher reproducibility and to equip fluid percussion injury machine (Dragonfly) in our laboratory. According to the method by Kita et al., central fluid percussion (maximum positive pressure 1000mg, maximum negative pressure 160mg) is supposed to be delivered through bone window on parietal region of Wister rats, making DAI with histologically confirmation. Unfortunately, we could not elicit the whole result of our project, we are planning to examine the expression of MSP mRNA and protein using this model and to perform transfection of the gene in ventricles with liposome, followed by analysis of the effect of suppression by siRNA.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2004 -2005 
    Author : 浅井 清文; 三浦 裕
     
    本研究課題では、アストロサイトの部位特異性を担う機能分子(アストロサイトの膜表面のレセプター、トランスポーター、チャネル、細胞接着因子類、あるいはアストロサイト産生性の神経栄養因子)の同定を行い、その発現量の差異が神経機能の調節にどのように関わっているか解明することを目的に研究を進めている。これまでに細胞培養法を用いて異なる脳内部位より培養したアストロサイト間で発現量に差異のある遺伝子を見いだしていたが、さらにvivoに近い系を用いるため、昨年度より迅速免疫組織染色法とlaser microdissection (LMD)法を用いて凍結切片から脳内部位別にアストロサイトを採取し、遺伝子発現をDNA microarrayにて比較することを開始した。8週齢の雄Wistar ratより脳全体を取り出し、ブレインマトリックスでスライスした後OCTコンパウンドで包埋し、6μmの凍結切片を作製した。一次抗体に抗GFAPを用い、ニチレイ、シンプルステインキットにて染色した。染色後、LMDにて大脳皮質と線条体から1000個ずつアストロサイトの細胞体部分を切除し、RNAを抽出、DNA microarrayにて比較を試みた。この方法で、これまでに、大脳皮質と線条体間で、数十の遺伝子に2倍以上の発現の違いが見いだされ、現在、in situ hybridization等による確認を行っている。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2003 -2005 
    Author : KATSUYA Hirotada; SOBUE Kazuya; ASAI Kiyofumi
     
    Aquaporins (AQPs) are water channel proteins, which increase water permeability via the plasma membrane and thus provide a route for rapid fluid movement. In the brain, AQP4 is abundantly expressed, and is highly concentrated in astrocyte membranes that are in direct contact with capillaries and pia. AQP4 is considered a major pathway for massive water shift across the plasma membrane under various conditions and is involved in brain edema formation. However, little is known concerning the mechanisms by which AQP4 expression is regulated in the brain. We investigated molecules, which interact with AQP4 and discovered RIL as a binding protein. Actually, it is confirmed that RIL directly interacted with AQP4 by in vitro binding assay, pull down assay, co-immunoprecipitation, and immunohistochemical staining. We are preparing a vector for a RIL null mouse, which reveals the regulation mechanisms of AQP4 by RIL. It is possible that RIL is a target molecule for a new therapeutic method of brain edema.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2003 -2004 
    Author : 大塚 隆信; 永谷 祐子; 浅井 清文
     
    整形外科疾患では種々の病態により関節水腫の発症をみる.いわゆる"水がたまる"といった症状であるが,疼痛を伴わない場合でも,関節可動域の制限など苦痛を伴う症例がある.日常臨床においては,関節穿刺,副腎皮質ホルモンの関節内投与などの対症療法がとられることが多く,しばしば再発する.関節水腫をきたす個々の疾患たとえば関節リウマチ,変形性関節症などの疾患解明は著しい進展をしているものの,関節水腫そのものの発生機序の分子機構の解明にはあまり進展はない.本研究では変形性膝関節症(OA)患者,関節リウマチ(RA)患者の滑膜組織,外傷後の関節水腫患者の滑膜組織の免疫組織染色を比較検討することと,in vivoの実験系にて,滑膜組織から得られた滑膜培養細胞を用いて,アクアポリン発現の誘導因子,抑制因子を明らかにすることを目的としている. 本年度は,昨年度に続き関節水腫をきたす各疾患の滑膜組織の免疫組織化学染色を行った.すなわちRA, OA患者の人工膝関節置換術,膝関節鏡視下手術のさいに患者の承諾を得て採取した滑膜組織を抗アクアポリン-1,7,8,9抗体を用いて,免疫組織化学染色した.水腫の著明な症例では,滑膜表層細胞にアクアポリン-1の高発現を認めた.この発現は水腫を引き起こすために発現しているのか,あるいは改善するために発現しているのかは明らかでないため、この分子機構を解明する必要がある。現在さらに滑膜培養細胞を用いて,RT-PCR法によりアクアポリン-1 mRNA発現を判定量的に測定し,さらにアクアポリンの誘導因子,抑制因子を明らかにする予定である.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2003 -2004 
    Author : ASAI Kiyofumi; MIURA Yutaka
     
    1, Analysis of promoter activity of the human glia maturation factor-gamma gene We determined the organization of the 9.5-kb hGMFG gene and characterized its promoter activity. The 5'-flanking region of the first exon has no TATA or CAAT boxes within a 226-bp sequence upstream from the initiation codon. Primer extension analysis and 5'RACE (rapid amplification of cDNA 5' ends) identified multiple transcription initiation sites within the region -84 to -70 nucleotides from the first ATG codon in a Kozak consensus sequence. A core promoter region was determined by transfecting a series of deletion constructs with a dual luciferase reporter system into rat astrocyte-derived ACT-57 cells. 2, Development of two-site enzyme immunoassays (EIA) for glia maturation factor beta (GMFB) and gamma (GMFG) We developed sensitive and specific two-site enzyme immunoassays (EIA) for glia maturation factor beta (GMFB) and gamma (GMFG) using specific antibodies raised in rabbits. These assay systems enabled us to identify GMFB and GMFG (GMFs) in both human and rat samples and they were used to investigate the tissue distribution and serum concentrations of human and rat GMFs. In the case of rat, relatively high levels of GMFB were found in the central nervous system, except for the spinal cord, and in thymus and colon. Higher levels of GMFG were found in the thymus, spleen and colon. The distribution of GMFs in human was similar to that in rat. In the rat, the maximum serum concentration of GMFG was at 4 weeks of age. The decrease in its level was rapid for the first 30 days of life in both sexes. On the other hand, the concentration of GMFB in serum did not change significantly with age. Similarly, in human, the concentration of GMFG in serum was highest in the 21-30-year-old group and began to decrease rapidly in the 30-year-old group. In contrast, the concentration of GMFB did not change significantly during this period. No significant sex differences in the serum levels of GMFs were observed in human and rat. 3, Analysis of expression of glia maturation factor beta after cryogenic injury We investigated the expression of GMFB during 56 days after cryogenic brain injury, using immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and enzyme immunoassay. Immunohistochemical analysis demonstrated that the GFAP-positive astrocytes around the lesion expressed GMFB protein, peaking 14 days after injury. Weak astrocytic expression of GMFB immunoreactivity was seen in sham-operated animal brains. Cryogenic injury (CI) induced GMFB mRNA in the lesioned side after 7 days with a maximum at 14 days. Western blotting revealed the induction of GMFB protein starting 1 day after injury, and continuing until 14 days after injury. In the enzyme immunoassay, GMFB protein concentration peaked 14 days after injury in extracts from the injured side of the brain, whereas in serum it peaked 1 day after injury. These data indicate that the expression of GMFB increased in the astrocytes around the lesioned area after cortical cryogenic brain injury.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2002 -2003 
    Author : NAGAYA Yuko; ASAI Kiyohumi; OTSUKA Takanobu
     
    Neovascularization, proliferation of synovial cells, and mononuclear cell influx and activation are characteristic events observed in synovial joints in the pathohistology of rheumatoid arthritis (RA). We have previously measured the concentration of the angiogenic factor, gliostatin (GLS), in sera and synovial fluids of RA patients, and demonstrated for the first time an enormously high concentration in RA synovial fluids as well as in RA sera. Furthermore our previous study demonstrated that GLS acts as a cytokine to augment its own synthesis in fibroblast-like synoviocytes (FLSs) obtained from patients with RA through an autocrine mechanism. It should be noted that GLS additionally caused induction and extracellular secretion of matrix metalloproteinase (MMP)-1 and MMP-3 triggering cartilage degeneration. Recently we reported that intraarticular injection of rHuGLS to rabbit knees induced RA-like synovitis. The purpose of this study was to investigate whether disease modified antirheumatic drugs (DMARDs) is involved in the regulation of GLS expression. FLSs were cultured and stimulated interleukin (IL)-1beta with or without DMARDs. Expression levels of GLS were determined by reverse transcription-polymerase chain reaction methods and enzyme-linked immunosorbent assays. In cultured rheumatoid FLSs GLSmRNA were found to be markedly increased by stimulation of IL-1beta. GLSmRNA in IL-1beta-stimulated FLSs were reduced by aurothioglucose and dexamethasone in a dose dependent manner. Methotrexate and sulfasalazine had no major influence on GLS production. These findings indicate that aurothioglucose and dexamethasone are responsible for the anti-rheumatic activity mediated via inhibition GLS production.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2001 -2003 
    Author : TAKEUCHI Akinori; ASAI Kiyofumi; SOBUE Kazuya
     
    Aquaporins, water channels, are a family of water-selective transporting proteins, that play an important role in water transport in the brain. Firstly, the expression of AQP family was investigated thoroughly in the central nervous system. In the brain, we detected the transcripts of AQP3, AQP4, AQP5, AQP8, AQP9 in astrocytes using the reverse transcription-PCR. These results suggest that AQP in astrocytes have roles in not only water transport but also other unknown functions. Secondly, regulatory mechanisms of AQP expressions were also investigated. The expression of AQP4 was down-regulated by protein kinase C (PKC). The expression of AQP5 was down-regulated by protein kinase A (PKA). The expression of AQP9 was up-regulated by PKA and down-regulated PKC. The p38 mitogen-activated proteinkinase (p38 MAPK) regulated the expression of AQP4 and AQP9. These results suggest that the expressions of AQPs are intricately regulated according to the conditions. Third, we examined the changes of AQP expressions under hypoxic stress. The expressions of AQP4 and AQP9 were up-regulated by hypoxia, but precise mechanisms for their regulation are still unclear. Further studies are needed to clarify the mechanisms. New therapeutic method for brain edema may be developed from our basic data. The agent of regulation of AQP expression or AQP antibodies might be new medicines for brain edema.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 2001 -2002 
    Author : 伊藤 彰師; 浅井 清文
     
    本研究は、麻酔薬によって中枢神経系で誘導、あるいは抑制される分子を、DNAマイクロアレイを用いて効率よくスクリーニングしようとするものである。平成13年度は、比較的培養が容易なアストロサイトを静脈麻酔薬であるプロポフォールで刺激した際に誘導あるいは抑制される分子の検索を行った。培養アストロサイト単独とプロポフォールで刺激したアストロサイトからmRNAを単離し、DNAマイクロアレイを施行した。その結果、プロポフォールによりmRNA発現が増加する分子が41種類、減少する分子が44種類検出された。 平成14年は、増加する分子群のうちHeat shock protein(HSP)に注目して詳細な検討を行った。HSPは、ストレス反応性タンパク質や抗アポトーシス作用を持つ分子群の転写を上昇する細胞内電子伝達系関連分子であり、プロポフォールの細胞保護作用の機序を説明できる可能性がある。主なHSPについてPCRによるアストロサイト内のmRNA発現定量法を確立し、プロポフォールによるHSP mRNA発現の増加、タンパク質発現の増加を調査中した。HSP84はプロポフォールにより、発現が増加する可能性があり、今後も検討を続けていきたい。 また、発現が減少する分子は多彩であるが、興味深いのは細胞骨格に関わる分子群が他種類検出されており、プロポフォールがアストロサイトの分化あるいは形態変化をモジュレイトしている可能性がある。なかでも、シンデカンは反応性アストロサイトにおいて高発現しており、脳損傷後の修復過程におけるグリオーシスに関与している。プロポフォールはシンデカンの発現を低下させることが分かり、今後脳損傷後のグリオーシス予防に使用できる可能性がある。 以上のように、プロポフォールの細胞保護作用の機序解明やプロポフォールの新しい作用の発見につながる研究の発端となった。本研究は新たな研究への萌芽的な機能を十分に果たしたと考える。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2001 -2002 
    Author : YAMADA Kazuo; SHIMADA Syoichi; AIHARA Noritaka; MASE Mitsuhito; ASAI Kiyofumi
     
    To detect changes in channel transporter, we used models of cerebral ischemia, cold-induced edema, subarachnoid hemorrhage (SAH) and intracerebral hemorrhage in rat. The transporters we detected are aquaporin type 1, 4,5 and 8, and myoinositol osmolyte transporter. In the cerebral ischemia, aquaporin type 4 mRNA is expressed in 3 to 7 days after ischemia, and aquaporin type 4 protein is increased in soma and disappearing from vascular foot, indicating turnover of aquaporin 4 is occurring. Similar findings are detected in the cold-induced edema model, indicating that aquaporin type 4 is functioning for resolution of brain edema. On the other hand aquaporins 1,4,5 and 8 is expressed in the cultured astrocyte exposed to hypoxia, indicating multipotential nature of aquaporins. Myoinositol transporter, one of the osmolyte transporters, is expressed in the neurons facing subarachnoid blood of SAH model, indicating osmotic pressure changes in SAH. Myoinositol transporter mRNA is also expressed in the neurons exposed to transient ischemia after SAH, indicating osmolyte changes in cerebral ischemia. We have detected cell death in substantial nigra after striatal hemorrhage in rat. During this process nigral neurons expressed myoinositol mRNA expression, which may relate to cell death. In Summary, channel transporter plays important role in brain injury and its recovery. Treatment trial must be focused in this area.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 2000 -2002 
    Author : ASAI Kiyofumi; MIURA Yutaka
     
    (1) Production of Specific Antibodies against Glia Maturation Factor beta (GMFB) and gamma (GMFG). Recombinant proteins of rat GMFB and GMFG were expressed by E. coli (BL21(DE3)) using pAED4 expression vector, and purified by column chromatography. Polyclonal antibodies were obtained by immunization to New Zealand White Rabbits. Specific antibodies were isolated by affinity chromatography. By western blot, no cross-reactivity was detected between GMFB antibody and GMFG, or between GMFG antibody and GMFB. (2) Establishment of Enzyme Immunoassay System for GMFB and GMFG. Sandwich EIA system was established by using the specific antibodies against GMFB and GMFG respectively. These EIAs enabled us to identify immunoreactive GMFB and GMFG in human plasma and rat organs. GMFB was expressed in brain, spleen, thymus, lung and intestine, and GMFG was expressed in thymus and spleen. These results correspond to the results of western blot and immunohistochemical analyses. (3) Cloning of genomic DNA of human and mouse GMFG. Genomic DNA of human and mouse GMFG were cloned. Sequence analyses revealed that both human and mouse GMFG gene were consisted by 7 exons.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1998 -2001 
    Author : ASAI Kiyofumi; MIURA Yutaka
     
    Our aim of this research was to identify the molecules that exhibit the regional heterogeneity of astrocyte. (1) Regional heterogeneity of the expression of neurotrophic factors in astrocyte. Astrocytes were cultured separately from cortex, hippocampus, olfactory bulb, cerebellum and spinal cord of rat embryo (E18). Total RNA was isolated and subjected to northern blot analysis. Glia maturation factor-beta was expressed ubiquitously. Expression of glia maturation factor-gamma was higher in cerebellum and spinal cord than other regions. High expression of lipocortin 1 was detected in hippocampus, cortex and cerebellum. (2) Analysis of regional heterogeneity of the astrocytic gene expression by Atras Array. Astrocytes were cultured separately from cortex, hippocampus and cerebellum of rat embryo (E18). Total RNA was isolated and subjected to analysis of Atras Array (Clontech). When the gene expression of cortex was compare with that of hippocampus, expression of 7 genes was higher in cortex than hippocampus, and 4 genes were higher in hippocampus than cortex. When compare cortex and cerebellum, 5 genes were higher in cortex, and 4 genes were higher in cerebellum. The expressions of Id-1, HSP27 and lipocortin 1 were confirmed by RT-PCR. The results from RT-PCR were consistent with the data from Atlas Array.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1999 -2000 
    Author : ASAI Kiyofumi; MIURA Yutaka
     
    (1) Establishment of immortal bovine brain endothelial cell To establish immortal brain endothelial cell, pSV3-neo which contains SV40 T antigen was introduced to primary cultured bovine brain endothelial cells. After selection by G418, we obtained six clones that exhibited the spindle-shaped appearance of normal brain endothelial cell. One of them, t-BBEC-117, retained the brain endothelial cell phenotype. (2) Establishment of immortal rat astrocyte pSV3-neo was introduced to rat primary cultured astrocytes. We obtained four immortal colony, which exhibited glial fibrillary acidic protein. A single clone, ACT-57, exhibited stable growth in a chemically defined medium and a stronger expression of NGF mRNA than that of normal astrocyte. (3) Identification of astrocytic factor which induce blood-brain barrier phenotype. Astrocyte in co-culture was found to tighten the intercellular contacts of t-BBEC-117. Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes. These data suggest that bFGF is one of the most plausible astorcytic factors to induce the blood-brain barrier properties of immortal brain endothelial cells.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1998 -2000 
    Author : MATSUI Nobuo; ASAI Kiyofumi; NAGAYA Yuko; OTSUKA Takanobu
     
    Neovascularization, proliferation of synovial cells, and mononuclear cell influx and activation are characteristic events observed in synovial joints in the pathohistology of rheumatoid arthritis (RA). The purposes of this study are to examine how gliostatin/platelet derived-endothelial cell growth factor (GLS/PD-ECGF) is involved in the molecular mechanism of cartilage degradation in RA with special reference to the GLS-induced gene expression and protein synthesis of matrix metalloproteinase (MMP)-1 (collagenase-1) and MMP-3 (stromelysin-1) and to examine synovial inflammation in rabbit knees induced by intraarticular administration of human GLS/PD-ECGF. GLS demonstrated a self induction of mRNA in cultured RA synoviocytes. GLS evoked a dose-dependent induction of MMP-1 and MMP-3 mRNAs and subsequently their extracellular secretions. Intraarticular injection of rHuGLS resulted in development of diffuse synovitis resembling rheumatoid arthritis. The mutant protein which was prepared by site-directed mutagenesis and lacking dThdPase activity also brought out the same effect. These findings suggest that GLS is a plausible pathogenic factor causing the extensive joint destruction in RA mediated via MMPs and that human GLS can cause RA-like synovitis in rabbit knee joints via a mechanism other than its thymidine phosphorylase activity.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1998 -1999 
    Author : ASAI Kiyofumi; KATO Taiji
     
    (1) Cloning of human GMF-gamma cDNA We isolated a human GMF-gamma cDNA from human fetal brain cDNA library. One of the clones contained a 573 nucleotide long insert and an open reading frame of 426 bp encoding a deduced protein of 142 amino acids. (2) Expression of human GMF-beta and GMF-gamma The cDNA of human GMF-beta and GMF-gamma were inserted to pAED4 expression vector and introduced to E. coli BL21(DE3). The expressed proteins were purified and subjected to antibody production. (3) Production of anti-human GMF-beta and GMF-gamma antibodies Recombinant human GMF-beta and GMF-gamma were used as immunogens for the production of polyclonal antibodies. The each anti-serum recognized human GMF-beta and GMF-gamma respectively, but either of them did not react to rat GMF-beta or GMF-gamma. (4) Cloning of rat GMF-gamma cDNA We isolated a rat GMF-gamma cDNA from rat pulmonary artery cDNA library. One of the clones contained a 553 nucleotide long insert and an open reading frame of 426 bp encoding a deduced protein of 142 amino acids. (5) Expression of rat GMF-beta and GMF-gamma The cDNA of rat GMF-beta and GMF-gamma were inserted to pAED4 expression vector and introduced to E. coli BL21(DE3). The expressed proteins were purified and subjected to antibody production. (6) Production of anti-rat GMF-beta and GMF-gamma antibodies Anti-rat GMF-beta and GMF-gamma serum were produced by the same way of human GMF-beta and GMF-gamma. The anti-sera recognized rat GMF-beta and GMF-gamma respectively.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1996 -1996 
    Author : 浅井 清文
     
    研究方法 1、グリア細胞成長因子(GMF)の酵素抗体法による定量法の確立 リコンビナント蛋白に対するポリクローナル抗体を使用し、抗体固相化ビーズとβ-galactosidaseでラベルした抗体を使用したサンドイッチEIA法を確立を試みた。 2、ヒト・グリア細胞成長因子(GMF) cDNAのクローニング ラットGMF cDNAをプローブとして、ヒト胎児脳cDNAライブラリーをスクリーニングし、ヒトGMF cDNAのクローニングを行った。 3、グリア細胞成長因子のアストロサイトへの遺伝子導入 アストロサイトへ発現ベクターを使用しcDNAを導入し、GMFをover expressionしたグリア細胞株を樹立を試みた。 研究結果 1、定法に従いサンドイッチEIA法を確立を試みたところ、GMFリコンビナント蛋白に対しては測定できることが判明したが、測定感度が十分でなく実験に使用するまでには至らなかった。今後、さらに抗体価の高い抗血清を作成し、測定感度を良くする方針である。 2、スクリーニングの結果、4131bpのcDNAをクローニングした。(DDBJ Accession No. AB001106)。 3、発現ベクターpKCRにラットGMF cDNAをクローニングした。このベクターをリポフェクション法によりラット初代培養アストロサイトに導入しGMFをover expressionしたグリア細胞株を樹立をした。Over expressionの状態は、ノーザンブロットおよびウエスタンブロットにより、mRNAおよび蛋白質レベルの両方にて確認した。今後、この細胞株を用いてGMFの機能解析を行う予定である。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1996 -1996 
    Author : SUZUKI Osamu
     
    Alcoholism induces serious functional and pathological abnormalities in many organs, particularly in the central nervous system. However, the molecular mechanisms accounting for these effects have not been identified, because ethanol can affect many different receptors and transpoters on the cell membrane. Recent studies suggest ethanol can modify intracellular signal transduction systems. We have proposed NG108-15, neuronal cultured cells as a model, and we have found that cAMP system is involved in the induction of heterologous desensitization, an example of ethanol tolerance at a cellular level. In the present project, we have established NG108-15 cells expressing transmitter receptors by stable transfection to identify receptors of which signal transduction system could be modified by chronic ethanol. At first, we checked the effect of chronic ethanol on promotor activity. We chose CMV,RSV and SV40 promotors and assessed the changes of these activeties using receptor binding assays or CAT assay. In NG108 cells transiently expressing CAT,chronic ethanol activated the activities of all promotors. However, chronic ethanol treatment did not change the expression of A_1 or D_2 receptors or CAT in some stable transfectants. These finding suggest not only promotors, but also cis elements juxtaposing with inserted cDNA would affect the effect of ethanol on the gene expresion. In our previous study, adenosine A_2 receptor is attributed to the generation of heterologous desensitization by accumulating extracellular adenosine after chronic ethanol treatment, but A_1 receptor seems not to be involved in the induction of these phenomenon. Protein Kinases play important roles in the intracellular signal transduction system and the translocation of these kinases is accompanied with these activation. So we focused on the translocation of protein kinases, especially A kinase (PDA) and C kinase (PKC), induced by ethanol. In NG108 cells, ethanol translocated catalytic subunit of PKA from cytosol to nucleus, but RI,a regulatory subunit of PKA was not translocated. Also, ethanol translocated some isozymes of PKC in NG108 cells. We will perform further studies on those translocations by chronic ethanol.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1995 -1995 
    Author : 加藤 泰治; 浅井 清文
     
    「ニューロン・ニューロン間のシグナル伝達を支えるのはグリアである」といっても過言ではない。有髄神経のミエリン鞘(オリゴデンドロサイト、シュワン細胞)やアストロサイト上のニューロトランスミッター受容体刺激に伴う、アストロサイト間のギャップジャンクションを介したCa^<2+>の伝播など、アストロサイトの役割を無視してシグナル伝達の研究は成立しない。われわれは、これまでニューロン性細胞の増殖・分化機構を追求するなかで、ニューロンとアストロサイトの共存培養系でニューロンは著明な分化(神経突起伸長とシナプス形成)をとげることを見いだし、これらの作用はアストロサイトが産生するタンパク質性因子であることを確認した[Brain Res.622(1933)299-302,659(1994)169-178]。 昨年度の本重点研究の成果として、(1)細胞内カルシウムシグナリング法を用いたシナプス形成モデル系において、大脳皮質、海馬、中隔野、線条体より調製したニューロンとアストロサイトを同系、異系の組み合わせで共培養しそれぞれのシナプス形成能を測定したところ、脳の同じ部位から調製したアストロサイトとニューロンの共培養系でのシナプス形成が一番良くシナプス形成することを見いだした。さらに(2)アストロサイト細胞内のCa^<2+>の動きを修飾する薬剤(IP_3レセプター拮抗薬やアストロサイトのギャップジャンクションの阻止薬)を用いて、短期と長期薬剤刺激によるシナプス形成の変化を検討した結果、シナプス形成にあずかるニューロンの細胞内Ca^<2+>の同期にはIP_3レセプターを介した細胞内Ca^<2+>の変動と、フィーダーアストロサイト間のギャップジャンクションを介したCa^<2+>の伝播が重要であることを確認した。またこの薬剤の長期投与によりシナプス形成能も阻害されることがわかった。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1995 -1995 
    Author : 加藤 泰治; 浅井 清文
     
    「アストロサイト由来神経栄養因子によるニューロン・グリア相関」をメインテーマとして研究する過程で、ニューロンおよびグリア発生に密接に関連すると思われるタンパク質性因子を同定した。なかでもグリア成長因子(GMF)、グリア増殖制御因子(グリオスタチン)の2種の因子は、いづれもアストロサイトが産生する神経栄養因子で、GMFとグリオスタチンについては分泌シグナルを持たない細胞質タンパクであることを証明した。さらに昨年度、グリアが神経芽腫細胞の増殖を特異的に抑制するタンパク性の神経芽細胞増殖抑制因子(NGIF)を産生していることを見い出した。この因子はアストロサイトが産生する弱塩基性タンパク質で、、分子量60Kのホモダイマー構造(120K)をとる。さらにこのNGIFはラット大脳皮質ニューロンに対し、生存維持および突起進展作用といったいわゆる神経栄養因子活性をもつことがわかった。この知見は、神経芽腫細胞増殖抑制因子(h-NGIF)が特異性の高いサイトカインであり、脳発生や神経機能修復過程で作用すると考えられる新しい神経栄養因子であることを示している。 将来的な臨床応用を考慮して、これまでのラットからヒトに切り替え、ヒト星状膠腫細胞(NAC1)が産生するヒトNGIFを用いて実験を遂行中である。いまのところ、定法のカラムワークとともにサブトラクション法も用いてNGIFの化学構造の決定を急いでいる。得られた遺伝子構造をもとに組換え体ヒトNGIFを大量する。この新規のサイトカインを用い神経芽腫の治療をめざす。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1994 -1995 
    Author : KATO Taiji; ASIA Kiyofumi
     
    Gliostatin is an scidic protein purified from human neurofibroma as glial growth inhibitory factor by Asai and Kato. Lately, we have demonstrated that its nucleotide sequence is completely identical to that of platelet-derived endothelial cell growth factor (PD-ECGF), which was purified from human platelets. Gliostatin/PD-ECGF is polypeptides of Mr=100,000 with a homodimeric structure comprising two 50-kDa subunits, one of which contains 482 amino acids but not carbohydrate moiety. The amino terminus has no signal sequence which is necessary for a protein to be secreted out of cells [5]. However, it has been confirmed that gliostatin/PD-ECGF has varied biological actions, but lacked the secretory signal sequence, such as 1) chemotactic activity for endothelial cells in vitro, 2) angiogenic activity in vivo, 3) glial differentiative and neurotrophic activities including neuronal surviving activity and neuritogenic activity in vitro, and 4) arthritogenic activity in vivo. Apart from much information of the molecular mechanism of its functions, little is known about the regulation of gene expression as well as the human tissue distribution and content of gliostatin/PD-ECGF in detail. And it is also not clear yet whether gliostatin/PD-ECGF lacking a signal sequence is extracellularly secreted in physiological conditions or not. Therefore, we have currently investigate the tissue-specific distribution of gliostatin/PD-ECGF by sensitive enzyme immunoassay (EIA), and the induction of its protein synthesis and gene expression with relevance to its secretion, using human tumor cell lines. Gliostatin/PD-ECGF was found to distribute in rather ubiquitous than specific human tissues and organs, with a relatively high levels in the tissues of digestive system (esophagus and rectum), brain, spleen, bladder and lung, but not in gall bladder, aorta, muscle, fat and kidney. Certain tumor cells (A431 and MKN74) appeared to release it into the conditioned medium. Expression of gliostation/PD-ECGF in epidermoid carcinoma cell (A431) and stomach cancer cell (MKN45) was induced by dibutyryl cyclic AMP and phorbol ester, and uniquely in MKN45 by hydrocortisone. In particular, this hydrocortisone specifically saused an increase of the apparent secretion of MKN74 without its cytotoxic effects, suggesting a possible secretion of gliostatin/PD-ECGF in the restricted but not universal cell line.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1994 -1994 
    Author : 加藤 泰治; 浅井 清文
     
    脳神経系での「液性因子によるニューロン・グリア相関」の研究を進める中、当該研究者らはラットグリアがマウス神経芽腫細胞の増殖を特異的に抑制するタンパク性因子(神経芽腫細胞増殖抑制因子:r-NGIF)を多量に産生していることを見い出した。今年度はヒト神経芽腫細胞増殖抑制因子(h-NGIF)の精製を試みた。当該研究者らの研究室で樹立したアストロサイトーマ(NAC-1)の無血清条件培養液を大量に調製し、エコノQ、エコノCM、スーパーロース120カラムを用い、ヒト神経芽腫細胞(TGW)の細胞増殖抑制活性を基準にして精製を試みた。活性は第3のカラムステップで消失し、条件培養液からの精製は困難と判断し、平行して検討してきた分子生物学的手法に切り替えることとした。つまり増殖相に発現が多く静止相に少ないh-NGIF発現量の差を利用した、ディファレンシアルハイブリダイゼーション法あるいはディファレンシアルディスプレイ法を用いてh-NGIFcDNAをクローニングすることにした。現在、増殖相と静止相のNAC-1細胞からそれぞれのcDNAライブラリーを作成することができ、増殖相特異的に発現するcDNAクローンを選択中である。えられたクローンをすべて塩基配列決定するとともに、プラスミドcDNAを直接発現ベクターに組みかえて、大腸菌あるいはCOS細胞にトランスフェクトし、発現されるタンパクの細胞増殖抑制活性を基にh-NGIFcDNAをクローニングする方法も試してみる。現在の生物活性測定法(TGWの細胞増殖抑制活性)はタンパクの直接発現法でも十分クローニングできる感度を持ち合わせている。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1993 -1993 
    Author : 加藤 泰治; 浅井 清文
     
    「血液・脳関門(BBB)の選択的物質透過性を担う構造体は何か、血管内皮細胞なのか、アストロサイトなのか」についての研究は乏しい。当該研究者らは、これまでアストロサイトの増殖・分化機構の研究を進めるなかで、アストロサイトは隣接する細胞の性質を直接接触あるいは液性因子を介して変化させることを見いだした。BBB構造に於いても、アストロサイトとの接触が生体内にひろく存在する内皮細胞の物質透過性に特異性を付与するものと考えられる。そこで本研究はアストロサイトと内皮細胞を二重層に培養したBBBモデルを調整し、その選択的透過性の機構を明らかにすることを目的に研究を進めた。 1、アストロサイト、内皮細胞の調整:アストロサイトはラット大脳よりタイプ1アストロサイトを、申請者(加藤)の開発した方法に従い、初代培養(1週間)から2次培養(5日間)したものを用いた。血管内皮細胞はウシの大動脈および大脳皮質より、コラゲナーゼ処理後、ナイロンメッシュに通して内皮細胞を単離培養した。とりわけウシ大脳皮質の内皮細胞は、収率は良くないが実験に供することができた。 2、BBBモデルの設計:交付申請者の方法にしたがって調整した二重層培養系をセットした。電顕的には二重層培養した内皮細胞に特異的にBBB固有のタイトジャンクションが観察された。 3、物質透過性実験:二重層培養系での物質透過性を動力学的解析は、複雑になるため二重培養系チャンパーを用いて内皮細胞をアストロサイトを分離した培養系を用いて透過性を検討した。とりあえずL-Glucoseを用いた結果では、大脳皮質由来内皮細胞に特異的透過性が認められた。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1993 -1993 
    Author : 加藤 泰治; 浅井 清文
     
    神経難病脳や神経変性疾患脳の神経細胞死は、何らかの神経栄養・発育因子の低下によるものかもしれない。当該研究者らはこれまで液性因子によるニューロン・グリア相関の研究を進めるなかで、ヒトグリア性細胞が神経芽細胞の増殖と分化を促進する新しい神経発育因子(グリオスタチン)を産生していることを見い出した。この因子は分子量50kの酸性タンパク質で、正常神経芽細胞の著しい突起伸展作用ばかりでなく、グリア増殖を抑制する作用を保持している。そこで今回の研究では、(1)グリオスタチンの分泌機構と遺伝子発現調節機構、(2)グリオスタチン反応性ニューロンおよびグリアのスクリーニング、などを行なった。 1、グリオスタチンの分泌機構:グリオスタチンに対するモノクローナル抗体を用いて、別に調製したエンザイムイムノアッセイ系〕で各種ヒト培養細胞のグリオスタチン分泌能をみると、数種の細胞がグリオスタチンを大量に細胞外に分泌していることが分かった。この分泌性グリオスタチンと神経線維腫由来のグリオスタチンの塩基配列には、これまでのところ大きな差異は認められていない。ジブチルcAMPやフォルボールエステル(TPA)によりグリオスタチンmRNAが誘導されることがわかり、現在分泌起点での変化を検討中である。 2、グリオスタチン遺伝子の発現調節機構の解明:グリオスタチンはタイプ1アストロサイトで産生されることが証明された。さらに現在、発育過程あるいは虚血ラット脳でのグリオスタチンmRNAの発現をモニターするために、ラットグリオスタチン(全長)cDNAをクローニング中である。 2.グリオスタチンに反応する正常神経芽細胞の分類:胎児ラット大脳皮質神経細胞のほか、現在、海馬、中隔野あるいは線状体より神経芽細胞を培養しグリオスタチン反応性を確かめている。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1992 -1992 
    Author : 加藤 泰治; 浅井 清文
     
    内皮細胞とアストロサイトを共培養した脳血液関門(BBB)モデル系を作成し、この系を使用して、BBB形成におけるアストロサイトやその他の因子の影響、及び各種物質、薬剤のBBB透過性を検討した。内皮細胞にはウシ大動脈内皮細胞、アストロサイトには胎生ラット脳のものを使用し、二重細胞培養の内側メンブレンの片面にアストロサイト、対面に内皮細胞を培養した。この内側メンブランを縦にセットして透過実験を行ッた。今回は透過物質にドーパミンを使用し、BBBのバリア機能の発現について検討した。すなわち、血管内腔側に相当するルミナールチャンバーに高濃度のドーパミンを入れ、時間を追って血管外側に相当するダブルニナールチャンバーに透過してくるドーパミンの濃度を電気化学検出器をディテクターとするHPLCによって測定した。 内皮細胞単独の場合と、アストロサイトと共培養した場合とで、内皮細胞のバリア機能に差が生じうるかどうかについて検討したところ、10%FCSを含んだ培地条件ではほとんど差が認められなかったが、1%FCS下では透過性に差が見られた(この時共培養系の透過実験の際にはアストロサイトをはがして、内皮細胞のみとして実験している)。またアストロサイトとの共培養時には電顕にて内皮細胞間にタイトジャンクションの形成が認められた。この結果より、大動脈内皮細胞に対しても、アストロサイトが内皮細胞のタイトジャンクション形成促進や物質透過性の減少に働く可能性が示唆された。今後はまずこれらの再現性を綿密にチェックし、また、脳毛細血管の内皮細胞も使用して同様の実験を行い、大動脈内皮細胞との比較を試みる予定である。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1992 -1992 
    Author : 加藤 泰治; 浅井 清文
     
    近年の分子生物学的手法の発達により、神経系の発生・分化調節、生存維持、という各段階においてグリア細胞から分泌される神経栄養因子によってニューロンの分化調節や生存維持が図られていることが次第に明らかにされてきている。また、脳損傷後の修復過程でも、反応性アストロサイトから分泌される神経栄養因子が重要な役割を果たしていることが明かにされている。このような状況のなかで、グリア細胞の増殖・分化がどのように調節されているかを明らかにすることが、ひいては、神経系の発生・分化調節、生存維持、脳損傷後の修復過程を理解することにつながることと考えられる。 我々が、神経線維腫より精製したグリア細胞増殖抑制活性を持つ蛋白性因子グリオスタチンは、アミノ酸配列の検索の結果、血小板由来内皮細胞増殖因子(PD-ECGF)とほぼ同じと考えられた。また、我々は、胎盤よりPD-ECGFを精製し、神経線維腫より精製したグリオスタチンと、グリア細胞増殖抑制活性、血管内皮細胞増殖促進活性を比較したところ、グリオスタチン、PD-ECGFともほぼ同じ活性を示し、さらに我々の作製したモノクローナル抗体でどちらの活性とも阻害された。以上の結果より、グリオスタチンはPD-ECGFとほぼ同一因子と考えられた。さらに、本年度の研究により、グリオスタチンが新しい神経栄養因子であることを見いだした。このグリオスタチンの多彩な中枢神経系での役割を考えると非常に興味深い。つまり、脳発達過程でグリオスタチンは、グリア、ニューロン、血管内皮細胞にそれぞれ異なった作用を発揮しながらニューロン機能を支えている。また、脳損傷後の組織修復過程において、グリオスタチンは損傷後の反応性グリオーシスの制御に関与している可能性もあると思われる。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1991 -1991 
    Author : 加藤 泰治; 浅井 清文; 杉山 成司; 小林 正紀; 木戸内 清; 和田 義郎
     
    1、当初の計画したBBBモデルの設定方法1に従い、アストロサイトおよびウシ大動脈内皮細胞は、それぞれ単独に培養細胞を調製できた。しかし方法2のBBBモデルの設定段階で、内皮細胞は問題なく内側チャンバ-の膜面で単相に培養できたが、アストロサイトは培養が難しいことがわかり、ゼラチン、フィブロネクチン、コラ-ゲン、および他の細胞外マトリックスを用いて膜面接着性を検討した結果、タイプ1コラ-ゲンがアストロサイトの膜面接着に一番適していることが分かった。 2、方法3の物質透過性実験にあたり、2つのBBBモデルを作成することにした。すなわち、(a)当初の計画どおり内側チャンバ-のバイオポア膜をはさんで2種の細胞を上下に培養するサンドイッチモデルと、(b)膜面の片側(内側)にアストロサイトと内皮細胞をマクラ状に培養したモザイクモデルを調製することができた。 また、実際のダブルチャンバ-を用いた自動潅流実験系の予備実験結果より、内外二つのチャンバ-を水平に設置した開放系より、内側チャンバ-を垂直に置いて左右別の閉鎖チャンバ-をそれぞれ同期に自動潅流するシステムが、定量的解析を行うには一番適していることが分かった。次年度はこの系を用いて、各種薬剤の選択的透過性実験を行う予定である。 3、アストロサイト由来のグリア細胞増殖抑制因子(グリオスタチン)が、アストロサイトの増殖抑制作用だけではなく、ニュ-ロンに対して生存維持と神経突起伸展作用を示し、さらに血管内皮細胞の増殖を修飾することを見いだした。現在、この因子の化学構造を解析中である。次年度は、この因子を含めて、アストロサイトが産生する各種の因子で内皮細胞をプライムし物質の選択的特異性発現に与える効果を検討する計画である。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1991 -1991 
    Author : 加藤 泰治; 浅井 清文
     
    当該研究者らは、これまでアストロサイトの増殖・分化機構の研究を進めるなかで、アストロサイトは隣接する細胞の性質を直接触あるいは液性因子を介して変化させることを見いだした。BBB構造に於いても、アストロサイトとの接触が内皮細胞の物質透過性に特異性を付与するものと考えられる。そこで本研究はアストロサイトと内皮細胞を二重層に培養したBBBモデルを調整し、さらに、このモデルを利用し、BBBの構造と機能を総合的に研究する。 1、アストロサイトおよびウシ大動脈内皮細胞は、それぞれ単独に培養細胞を調整できた。しかし方法2のBBBモデルの設定階段で、内皮細胞は問題なく内側チャンバ-の膜面で単相に培養できたが、アストロサイトは培養が難しいため、細胞外マトリックスを用いて膜面接着性を検討した結、タイプ1コラ-ゲンがアストロサイトの膜面接着に一番適していることが分かった。 2、方法3の物質透過性実験にあたり、2つのBBBモデルを作成することにした。すなわち、(a)当初の計画どおり内側チャンバ-のバイオポア膜をはさんで2訊の細胞を上下に培養するサンドイッチモデルと、(b)膜面の片側(内側)にアストロサイトと内皮細胞をマクラ状に培養したモザイクモデルを調製することができた。 また、実際のダブルチャンバ-を用いた自動潅流実験系の予備実験結果より、内外二つのチャンバ-を水平に設置した開放系より、内側チャンバ-を垂直に置いて左右別の閉鎖チャンバ-をそれぞれ同期に自動潅流するシステムが、定量的解析を行うには一番適していることが分かった。次年度はこの系を用いて、各種薬剤の選択的透過性実験を行う予定である。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1991 -1991 
    Author : 加藤 泰治; 浅井 清文
     
    神経難病や神経変性疾患の神経細胞死は、何らかの神経発育因子(NTF)の低下あるいは過剰産生によるものかもしれない。とくにニュ-ロンを保護するアストロサイトや軸索を取り巻くシュワン細胞からニュ-ロンに作用する多くのNTFが産生されていることが分かってきた。研究者は、シュワン細胞起源のヒト神経線維種がニュ-ロン性細胞(ヒト神経芽腫細胞、ラット皮質ニュ-ロン)の増殖あるいは、分化を促進する2種類の新しいNTFを産生していることを見いだした。その一つは神経芽腫細胞の増殖促進活性をもつNBGFで、化学的にはウリジンあるいはアデノシン誘導体であった(論文投稿中)。他の一つはグリアの増殖抑制作用とNTF活性を併せ持つグリア細胞増殖抑制因子(GGIF:グリオスタチン)であることを証明した。とりわけ本年度は、グリオスタチンを中心に研究を進め、以下の結果を得た。神経線維腫から5種類のカラム(ブル-トヨパ-ル、DEAEセファセル、ブチルト-ヨ-パ-ル、ハイドロキシルアパタイト、MonoQカラム)を用いて精製したグリオスタチンは、SDSーPAGE上では、分子量の50kの単一バンドとして泳動された。グリオスタチンはグリアによって産生され、グリア自身に作用するいわゆるオ-トクリン形成で作用するユニ-クな因子である。さらに、大脳皮質ニュ-ロンの生存維持や神経突起伸展作用を併せもつことは、脳神経の発育過程や神経機能修復過程で重要な機能を果たしていることを予想させるだけでなく、グリオスタチンの将来的な臨床応用(脳腫瘍、神経疾患の治療)を考える上で、重要な知見である。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1991 -1991 
    Author : 加藤 泰治; 浅井 清文
     
    研究者は、von Recklinghausen氏病患者より得られた神経線維腫(neurofibroma)中に、脳腫瘍(アスロトサイト-マ、グリオブラスト-マ、オリゴデンドログリオ-マなど)の増殖を抑制するタンパク質性因子(グリア細胞増殖抑制因子:GGIF)が存在することを見いだした。今年度は、この因子を5種類のカラムクロマトグラフィ-を用いて完全精製した。さらにこの因子にニュ-ロンの再生機能を促進する新しい神経栄養因子(Neurotrophic factor:NTF)活性があることを見いだした。この因子はグリア細胞の増殖をサイトスタチックに抑制することから、われわれはこの因子をグリオスタチンと命名した。 神経線維腫から5種類(ブル-トヨパ-ル、DEAEセファセル、ブチルト-ヨ-パ-ル、ハイドロキシルアパタイト、MonoQカラム)のカラムを用いて精製したグリオスタチンは、SDSーPAGE上では、分子量50Kの単一バンドとして泳動された。ス-パ-デックス200によるゲル濾過法による推定分子量は100Kであったことより、グリオスタチンは通常ホモダイマ-構造をしているものと考えられる。グリオスタチンの生物作用は、動物種差を問わず、グリオ-マの増殖を特異的に抑制する。このグリオスタチンは、神経系ではグリアによって産生され、グリア自身に作用するいわゆるオ-トクリン形式で作用するユニ-クなサイトカインである。さらに、大脳皮質ニュ-ロンの生存維持や神経突起伸展作用を併せもつことは、グリオスタチンの将来的な臨床応用を考える上で、重要な知見である。つまり、脳腫瘍に対する治療薬(サイトカイン)として、あるいは脳外科手術後や脳損傷後の後遺症の原因となる反応性グリオ-シスを予防する薬剤としてグリオスタチンを用いることが、同時に損傷を受けた周囲ニュ-ロンの機能修復にも役立つことにつながることを意味している。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research
    Date (from‐to) : 1990 -1991 
    Author : KATO Taiji; ASAI Kiyofumi
     
    Two kinds of novel neural trophic factors were detected in von Recklinghausen neurofibroma (NF1) extracts. one of two, neuroblastoma growth factor (Mr<5 kDa), promotes the proliferation of human neuroblastoma cell and survival and neurite-extension of rat cortical neuron, but is distinct from nerve growth factor (NGF) or NGF-like factors. The other trophic factor is a glial growth inhibitor (Mr=100 k), which suppresses the growth of glioma cell lines, astrocytoma, glioblastoma, oligodendroglioma and Schwannoma. These factors do not appear to be previously identified cytokines or growth factors such as interleukins, granulocyte colony-stimu-lating factor, NGF and fibroblast growth factor, Ciliary neurotrophic factor-like activity was also detected in the extracts. Anovel endogenous polypeptide inhibitor of proliferation and DNA synthesis of glial cells, gliostatin, was purified from the extracts of neurofibroma by a procedure comprising column chromatography with Blue-Toyopearl and DEAE-Sephacel, and high-performance liquid chromatography with Butyl-Toyopearl, Hydroxylapatite and Mono Q. monoclonal antibody raised against partially purified (Hydroxylapatite step) gliostatin adsorbed the growth inhibitory activity of gliostatin and immunochemically visualized the putative gliostatin bands on Western blot analyses, but showed no cross-reactivity with known cytokinesis. Although the product showed an apparent Mr=100 k accompanied by an inhibitory activity on Superdex 200 column chromatography, it migrated at a lower apparent Mr=50 k under the reducing condition on Western blotting, indicating that a homodimeric structure of native gliostatin consisted of 50 KDa subcomponents. Gliostatin was a potent growth inhibitor acting at nM concentration against all glial tumor cells d glia maturation factor (GMF)-stimulated astroblasts but not neuronal cells.
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1990 -1990 
    Author : 加藤 泰治; 浅井 清文
     
    アルツハイマ-病ばかりでなく各種の神経変性疾患者脳の神経細胞死は、何らかの神経栄養・発育因子(NTF)の低下あるいは過剰産生によるものかもしれない。とくに神経細胞を保護するアストロサイトや軸索を取り巻くシュワン細胞から神経細胞に作用する多くの液性因子(NGF、BDNTF,CNTF)が産生されることが知られている。研究者はこれまで液性因子によるニュ-ロン・グリア相関の研究を進めるなかで、ヒトグリア性細胞が神経芽細胞の増殖と分化を促進する新しい神経発育因子(NGPE)を産生していることを見いだした。 NGPFはフォンレックリングハウゼン氏病患者より得られた神経線維腫組織中に存在し、ヒト神経芽腫細胞(GOTO,TGW,NAGAI)に対し強いDNA合成を促進する活性を持つ。この因子は神経線維腫の抽出液より、熱(100℃、10分間)および酸(pH2.3)処理後、ゲル濾過(BioーGel Pー10、pH2.3)、逆相分取用カラム(LOPーODS)、陰イオン交換カラム(MonoーQ)、逆相系カラム(ODS)にかけ精製した。NGPFは熱・酸安定性の分子量4、400のペプチトで神経芽腫細胞に対し細胞増殖促進効果を示し、数nMでGOTO細胞のDNA合成を効約4倍に増強する。また正常ラット大脳神経細胞の神経突起伸長を促すが、ラット褐色細胞腫(PC12)には無効であった。化学構造的には、アミノ末端はブロックされているだけでなくペプチド鎖に直接他の化合物が結合している。現在、FABーMASやNMRを用いてNGPFの全構造を解析中である。mRNAを直接カエルの卵に刺入して、NGPFcDNAをクロ-ニングする操作を行ったが、ニ次クロ-ニングの段階で活性は同定出来なくなり、最初に得られたmRNAの自然分解のためと結論された。
  • 日本学術振興会：科学研究費助成事業
    Date (from‐to) : 1990 -1990 
    Author : 加藤 泰治; 浅井 清文
     
    グリア細胞成長因子(GMF)の発現細胞の検索は、これまでおもにラット由来のグリア性細胞(アストロサイトやその腫瘍性細胞)を中心に行われてきた。今年度になり、ヒトの脳腫瘍から5種類のアストロサイト-マおよびグリオブラスト-マを樹立することができた。これらの細胞はいずれも強いGMF産生能を有することが分かった。現在、これらの細胞の増殖能とGMF発現量との関係を、GMFcDNAプロ-ブを用いたmRNAレベル量の変化をもとに検討している。 昨年8月、われわれがウシ由来GMFのcDNAクロ-ニングに成功した時点で、アイオア大学のLimらはGMFの一次構造を発表した。この成果は、われわれの研究が遅れをとったという反省もあるが、反面、長年に渡って研究してきたこのGMFが、既知の成長因子とは全く異なる物質で、グリアのオ-トクリン増殖機構を担っている成長因子であるというわれわれの仮説を証明してくれたことに多大なよろこびを感じている。今後は、われわれが得たプロ-ブを用いてヒトグリオ-マの増殖に関わるGMFの発現調節機構を正常のアストロサイトのそれと比較してゆきたい。また最近、GMFの研究を進める過程で、GMFの細胞増殖促進作用を特異的に阻害するタンパク質性因子(GGIF)をグリア性細胞が産生していることを見いだした。この因子は腫瘍性グリア(アストロサイト-マ、グリオブラスト-マ)の細胞増殖を著明に抑制する新しいサイトカインであることも分かった。この相反する作用をもつGMFとGGIFは、ともにグリアで産生され、さらにグリア自身に作用するオ-トクリンタイプの調節機能を持つ因子である。今後はグリアの腫瘍性増殖発現と、この2つの因子のmRNAおよびタンパク質レベルでの発現との関係を上述した細胞を用いて検討する予定である。
  • 血液脳関門の機能とその形成機構の解明
  • アストロサイトによる 神経機能調節機構の解明
  • アストロサイトの機能解析
  • ニューロン・グリア相関
  • Neuron-Glia Interaction

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