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目的应用热休克蛋白90(Hsp90)过表达系统探讨Hsp90是否抑制肿瘤坏死因子α(TNFα)诱导的细胞凋亡及线粒体细胞色素c的释放。方法采用电穿孔技术建立稳定过表达Hsp90的细胞克隆,应用激光共聚焦显微镜和流式细胞仪观察TNFα和放线菌酮(CHX)诱导的细胞凋亡。应用W estern b lotting方法检测细胞色素c的变化。结果相差显微镜观察Hsp90过表达细胞与对照细胞相比悬浮细胞较少(分别为18%和41%),PBS冲洗后剩余黏附细胞较多。应用共聚焦显微镜进行TUNEL测定结果显示大部分对照细胞发生凋亡,相对较少的Hsp90细胞发生凋亡。W estern b lotting检测Hsp90细胞中细胞色素c的表达与对照组相比明显减少。结论Hsp90过表达抑制TNFα诱导的细胞凋亡及线粒体细胞色素c释放,提示其在凋亡信号传导通路线粒体水平发挥抑制作用。
Objective To investigate whether Hsp90 can inhibit tumor necrosis factor α (TNFα) -induced apoptosis and release of mitochondrial cytochrome c by using Hsp90 overexpression system. Methods Cell clones stably expressing Hsp90 were established by electroporation. The apoptosis induced by TNFα and cycloheximide (CHX) was observed by laser confocal microscopy and flow cytometry. The changes of cytochrome c were detected by Western blotting. Results The phase contrast microscopy showed that Hsp90 overexpressing cells had less suspended cells (18% and 41%, respectively) than the control cells, and there were more residual cells after PBS washing. Confocal microscopy TUNEL assay results showed most of the control cells apoptosis, a relatively small number of Hsp90 cells apoptosis. Western blotting showed that the expression of cytochrome c in Hsp90 cells was significantly reduced compared with the control group. Conclusion Hsp90 overexpression inhibits TNFα-induced apoptosis and mitochondrial cytochrome c release, suggesting that Hsp90 may play an inhibitory role at the mitochondrial level of apoptotic signal transduction pathway.