PI3K在肿瘤坏死因子诱导L929细胞程序性坏死过程中的调控作用研究

发布时间：2018-11-21 15:21

【摘要】：目的探讨磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinase,PI3K)在肿瘤坏死因子α(tumor necrosis factor alpha,TNFα)诱导细胞程序性坏死过程中的调控作用与机制。方法利用慢病毒介导的RNA干涉技术构建PI3K催化亚基p110α敲低、RIP1敲低及RIP1与p110α双敲低的L929细胞株,Western印迹或RT-PCR验证敲低效果。同时,利用Western印迹检测AKT和混合系激酶区域样蛋白(MLKL)磷酸化及MLKL多聚化。流式细胞术和显微拍照术检测细胞死亡。结果 PI3K和AKT抑制剂及p110α敲低都可降低AKT磷酸化水平,并阻断TNFα诱导的L929细胞程序性坏死。敲低RIP1并不抑制TNFα与Z-VAD联合诱导的L929细胞程序性坏死,但这种RIP1非依赖性细胞程序性坏死能为p110α敲低抑制。此外,p110α敲低可抑制TNFα与Z-VAD联合诱导的MLKL活化及其介导的细胞程序性坏死。结论 PI3K是调控细胞程序性坏死的重要靶点,可通过RIP1依赖及非依赖性方式激活,进而活化AKT与MLKL,启动TNFα诱导的细胞程序性坏死。
[Abstract]:Objective to investigate the role and mechanism of phosphatidylinositol 3-kinase (phosphatidylinositol-3-kinase,PI3K) in the process of programmed necrosis induced by tumor necrosis factor 伪 (tumor necrosis factor alpha,TNF 伪. Methods L929 cell lines with PI3K catalytic subunit p110 伪 knockdown, RIP1 knockdown and RIP1 and p110 伪 double knockout were constructed by lentivirus mediated RNA interference. Western blot or RT-PCR were used to verify the knock down effect. At the same time, AKT and mixed kinase-like protein (MLKL) phosphorylation and MLKL polymerization were detected by Western blotting. Cell death was detected by flow cytometry and microphotography. Results both PI3K and AKT inhibitors and p110 伪 knockout could decrease the level of AKT phosphorylation and block the programmed necrosis of L929 cells induced by TNF 伪. Low RIP1 knockout did not inhibit programmed necrosis of L929 cells induced by TNF 伪 and Z-VAD, but this RIP1 independent cell programmed necrosis could inhibit p110 伪 knockdown. In addition, p110 伪 knockout can inhibit the MLKL activation induced by TNF 伪 and Z-VAD and its mediated cellular programmed necrosis. Conclusion PI3K is an important target to regulate programmed necrosis of cells, which can be activated by RIP1 dependent and independent way, and then activate AKT and MLKL, to initiate TNF 伪 induced programmed necrosis of cells.
【作者单位】：安徽医科大学研究生学院;军事医学科学院基础医学研究所;
【基金】：国家自然科学基金资助项目(31201041) 国家重大科技专项重大新药创制资助项目(2014ZX09304313-003)
【分类号】：R329.2

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