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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。
Objective To investigate the regulatory effect and mechanism of phosphatidylinositol 3-kinase (PI3K) on the process of apoptosis induced by tumor necrosis factor alpha (TNFα). Methods The lentivirus-mediated RNA interference technique was used to construct p110α knockdown, RIP1 knockdown and double knockdown of RIP1 and p110α in L929 cell line. The knockdown effect was confirmed by Western blot or RT-PCR. At the same time, AKT and mixed kinase kinases (MLKL) phosphorylation and MLKL multimerisation were detected by Western blotting. Cell death was detected by flow cytometry and microscopy. Results Both PI3K and AKT inhibitors and p110α knockdown decreased AKT phosphorylation and blocked TNFα-induced apoptosis in L929 cells. Knockdown of RIP1 did not inhibit the apoptosis of L929 cells induced by TNFα and Z-VAD, but this RIP1-independent programmed cell death was inhibited by p110α knockdown. In addition, p110α knockdown inhibited MLKL activation induced by TNFα in combination with Z-VAD and its apoptotic cell-mediated necrosis. Conclusion PI3K is an important target of programmed cell necrosis. It can be activated by RIP1 in a dependent and independent manner, which in turn activates AKT and MLKL and initiates TNFα-induced programmed cell death.