Cell signaling of the neuroprotective cytokines

神经保护细胞因子的细胞信号传导

• 批准号: 13671440
• 负责人: HATA Ryuji
• 金额: $ 2.24万
• 依托单位: Ehime University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671440/
• 关键词: Stat; Cerebral Ischemia; Cell Death; Bcl-xL; Cytokine

Introduction : Recently, we reported that Stat3 was activated following cerebral ischemia in mouse [1]. However, little is known about the function of activated Stat3 in the ischemic brain. Here we demonstrate the increased expression of activated Stat3 promotes cell death in clutured astrocytes.Methods : Astrocytes from the forebrains of newborn rats were cultured as described [2] with some modifications. Recombinant adenovirus expressing Stat3 wild type (St3.Wt), Stat3 dominant negative type (St3.DN) and LacZ were constructed. Astocytes were exposed to adenovirus vector expressing Stat3.Wt, Stat3.DN or LacZ (multiplicity of infection 10) for 2h and incubated with a virus free fresh medium up to 72h. At 72h later, astrocytes were homogenized in lysis buffer and immunoblotted with antibody against Stat3, p-Stat3 (Tyr-705) and beta-actin. Cell death was quantified by LDH assay. Caspase-3 like activity was evaluated at 12h, 24 and 48h after the virus inoculation using a caspase-3 fluorometric protease assay kit.Results : Western blots analysis revealed feat Ad.St3.wt remarkably induced p-Stat3(Tyr-705) while Ad.St3.DN and Ad-LacZ did not. These results demonstrated that both Ad.St3.Wt and Ad.St3.DN induced Stat3 protein in cultred astrocytes and only Ad.St3.Wt b could phosphorylate Stat3 at Tyr-705 site and activate Stat3. LDH assay revealed that overexpression of St3.wt promoted cell death while overexpression of St3.DN and LacZ did not. In addition, caspase-3 like activity of the St3.Wt treated group was significantly increased than that of the St3.DN group at 2d after virus inoculation.Conclusion : Our Data have shown that over-expression of Stat3 promotes cell death. Activation of caspase-3 activity may be involved in this cell death mechanism. References : [1] Wen et al.; Neurosci Lett, 303:153-156, (2001), [2] Tanaka J et al.; Glia, 28:85-96 (1999)

引言：最近，我们报道了Stat 3在小鼠脑缺血后被激活[1]。然而，关于活化的Stat 3在缺血脑中的功能知之甚少。在这里，我们证明了活化的Stat 3表达的增加促进了培养的星形胶质细胞的细胞死亡。方法：如[2]所述，对新生大鼠前脑的星形胶质细胞进行了培养，并进行了一些修改。构建表达Stat 3野生型（St3.Wt）、Stat 3显性阴性型（St3.DN）和LacZ的重组腺病毒。将星形细胞暴露于表达Stat3.Wt、Stat3.DN或LacZ的腺病毒载体（感染复数10）2小时，并与无病毒的新鲜培养基孵育长达72小时。在72小时后，将星形胶质细胞在裂解缓冲液中匀浆，并用抗Stat 3、p-Stat 3（Tyr-705）和β-肌动蛋白的抗体进行免疫印迹。通过LDH测定定量细胞死亡。结果：Westernblot分析显示，Ad.St3.wt对p-Stat 3（Tyr-705）有明显的诱导作用，而Ad.St3.DN和Ad-LacZ对p-Stat 3（Tyr-705）无明显诱导作用。结果表明，Ad.St3.Wt和Ad.St3.DN均可诱导培养的星形胶质细胞表达Stat 3蛋白，只有Ad.St3.Wt B b能使Stat 3在Tyr-705位点磷酸化并激活Stat 3。乳酸脱氢酶（LDH）测定显示，St3.wt过表达促进细胞死亡，而St3.DN和LacZ过表达则无此作用。另外，在病毒接种后2d，St3.Wt处理组的caspase-3样活性显著高于St3.DN组。结论：我们的数据表明过表达Stat 3促进细胞死亡。caspase-3活性的激活可能参与了这种细胞死亡机制。参考文献：[1] Wen et al.; Neurosci Lett，303：153-156，（2001），[2] Tanaka J et al.; Glia，28：85-96（1999）

项目成果

Masumura M, Hata R, Uetsuki T, Nishimura I, Nagai Y, Sawada T: "In vivo gene transfer to cerebral white matter lesions with a recombinant adenovirus vector"Biochem Biophys Res Commun. 287. 440-444 (2001)

Masumura M、Hata R、Uetsuki T、Nishimura I、Nagai Y、Sawada T：“用重组腺病毒载体将基因转移到脑白质病变中”Biochem Biophys Res Commun。

Wen TC, Sadamoto Y, Tanaka J, Zhu PX, Nakata K, Ma YJ, Hata R, Sakanaka M: "Erythropoietin protects neurons against chemical hypoxia and cerebral ischemic injury by up-regulating Bcl-xL expression"J Neurosci Res. 67. 795-803 (2002)

Wen TC、Sadamoto Y、Tanaka J、Zhu PX、Nakata K、Ma YJ、Hata R、Sakanaka M：“促红细胞生成素通过上调 Bcl-xL 表达来保护神经元免受化学缺氧和脑缺血损伤”J Neurosci Res。

Hermann DM, Kuroiwa T, Hata R, Gillardon F, Ito U, and Mies G: "Expression of redox factor-1, p53-activated gene 608 and caspase-3 messenger RNAs following repeated unilateral common carotid artery occlusion in gerbils-Relationship to delayed cell injury

Hermann DM、Kuroiwa T、Hata R、Gillardon F、Ito U 和 Mies G：“沙鼠重复单侧颈总动脉闭塞后氧化还原因子 1、p53 激活基因 608 和 caspase-3 信使 RNA 的表达 - 与

Masumura M, Hata R, Akatsu H, Kosaka K, Yamamoto T, Nagai Y and Sawada T: "Increasing in situ nick end labeling of oligodendrocytes in white matter of patients with Binswanger's disease"J Stroke Cere Dis.. 1055-1062 (2001)

Masumura M、Hata R、Akatsu H、Kosaka K、Yamamoto T、Nagai Y 和 Sawada T：“增加 Binswanger 病患者白质中少突胶质细胞的原位切口末端标记”J Stroke Cere Dis.. 1055-1062（2001 年）

Wen T et al.: "Induction of phosphorylated Stat3 following focal cerebral ischemia in mice."Neurosci Lett. 303. 153-156 (2001)

Wen T 等人：“小鼠局灶性脑缺血后磷酸化 Stat3 的诱导。”Neurosci Lett。