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  

Identification of role of PI3K in mediating necroptosis of L929 cells induced by tumor necrosis factor alpha

作　　者：常喜喜 胡世平[2] 王宇[2] 王丽丽[2] 武帅[2] 王籽橙 杜芝燕[2] 于继云[2] 张毅[1,2] 陈国柱[2] 

机构地区：[1]安徽医科大学研究生学院,合肥230032 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《军事医学》2017年第1期25-32,共8页Military Medical Sciences

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)

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。Objective To identify the role of phosphatidylinositol-3-kinase（PI3K） in mediating necroptosis induced by tumor necrosis factor alpha（TNFα） and the involved mechanism. Methods Knockdown of p110α,receptor-interacting protein 1（RIP1） or both p110α and RIP1 was mediated by the specific short hairpin RNA（shRNA） lentivirus and verified by RT-PCR or Western blotting. In addition,Western blotting was used to detect phosphorylation of mixed lineage kinase domain-like protein（MLKL） and protein kinase B（AKT） or tetramerization of MLKL. Cell death was measured by microscopy and flow cytometry. Results AKT phosphorylation and TNFα-induced necroptosis of L929 cells were suppressed by the inhibitors of PI3 K or AKT,as well as p110α knockdown. Moreover,RIP1 knockdown did not inhibit L929 cell death induced by TNFα plus Z-VAD,but the RIP1-independent necroptosis was inhibited by p110α knockdown. In addition,p110α knockdown suppressed MLKL phosphorylation and tetramerization induced by TNFα with Z-VAD in L929 cells.Conclusion PI3 K mediates necroptosis of L929 cells induced by TNFα by activating AKT and MLKL,respectively.

关 键 词：磷脂酰肌醇3-激酶 肿瘤坏死因子Α 细胞程序性坏死 蛋白激酶B 混合系激酶区域样蛋白 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]