ERK通路对体外培养星形胶质细胞缺氧/复氧后TNF-α分泌的影响

Effect of ERK pathway on TNF-α secretion of astrocytes by hypoxia/reoxygenation

目的分析ERK通路对缺氧/复氧后反应性星形胶质细胞TNF-α分泌的影响,从而探讨ERK通路在星形胶质细胞反应性改变的可能作用机制,为临床研究提供理论支持。方法参照McCarthy方法星形胶质细胞(AC)原代培养,传至第3代,细胞自然纯化。将AC分为正常对照组(C组)、缺氧/复氧组(H/R组)、缺氧/复氧阻滞剂组(H/R+M组)。每组设缺氧4h、缺氧8h、复氧6h、12h、24h、48h6个时间点,建立AC缺氧/复氧模型。Western-blot法半定量分析T-ERK,P-ERK的表达情况,TNF-αELISA试剂盒测定细胞凋亡情况。采用SPSS18.0统计软件包进行数据分析。结果 (1)Western-blot:缺氧组较正常组相比ERK表达明显升高(P<0.05),阻滞剂组较无阻滞剂组p-ERK蛋白表达量显著下降(P<0.05)。(2)TNF-αELISA:缺氧后TNF逐渐升高,复氧48h时达高峰(P<0.05),加入ERK阻滞剂后升高更明显(P<0.05)。结论星形胶质细胞缺氧复氧后存在ERK通路的激活,ERK通路在星形胶质细胞缺氧损伤后反应中发挥生物学作用与TNF-α有关。

Objective To study the ERK pathway changes in reactive astrocytes（AC） after hypoxia and reoxygenation,and its effect on TNF-α secretion,and to provide a theoretical support for clinical research.Methods By the method of McCarthy,the cells were passaged in the third generation.The cells naturally purified.The AC was divided into the normal control group（C）,hypoxia/reoxygenation group（H/R）,hypoxia/reoxygenation-blocker groups（H/M＋R）.After eight-hour exposure to hypoxia,we started to reoxygenation time.The six-time point was settled as the hypoxia 4 hours,8 hours,and the reoxygenation 6 hour,12 hours,24 hours,48 hours.The Western-blot used to semi-quantitatively analyze the T-ERK,P-ERK expressions.The TNF-α secretion of astrocytes was measured by TNF-α ELISA kit.The SPSS18.0 statistical package was used for data analysis.Results ERK expression was significantly increased in the hypoxia group than normal（P0.05）.After given the blocker,the expression level decreased significantly than non-blockers（P0.05）.After hypoxia,TNF-α increased gradually and reached the peak at the point of hypoxia 48 hours（P0.05）.The group with ERK blocker has significant increase compared to no blockers（P0.05）.Conclusion Astrocytes exposed to hypoxia,it has the activation of the ERK pathway.ERK pathway plays an biological role correlated with TNF-α.