Bcl-xl阻断肿瘤坏死因子α诱导的caspase 8活化及细胞凋亡

目的探讨BCl-xl对肿瘤坏死因子α(TNFα)介导的细胞凋亡信号通路以及细胞凋亡的影响。方法将iκBα负性突变体质粒pmiκB与绿色荧光蛋白(GFP)表达质粒pEGFP-Cl，pmiκB、DBcl-xl／HA与pEGFP-Cl分别共转染HeLa细胞；将pBcl-xl／HA转染核因子κB(NFκB)／p65基因敲除的p65／MEF细胞，并用嘌呤霉素筛选及westernblot鉴定获得稳定表达Bcl-xl的细胞系p65／／Bcl-xlMEF。用TNFα处理HeI。a／miκB、IteLa／miκB／Bcl-xl细胞以及p65／MEF、p65／／Bcl-xlMEF细胞，并在冠微镜下观察细胞形态变化、台盼蓝染色计算死亡细胞以检测各组细胞凋亡情况；采用western blot检测凋亡信号通路中caspase8活化及腺苷二磷酸核糖聚合酶(PARP)裂解情况。结果单独转染pmiκB的HeLa细胞，TNFα诱导的细胞凋亡明显，表现为细胞变圆、脱落、死亡，而PBCl-xl／HA与PmiκB共转染的HeLa细胞，TNFα处理后未见细胞凋亡。P65／MEF细胞经TNIrα处理发生凋亡，该作用在TNFα处理后4h即可出现，处理12h后绌胞死亡率为90％，而在表达Bcl-xl的p65／／Bcl-xlMEF细胞，TNFα处理24h也未见细胞凋亡或死亡发生。Westernblot检测表明单独转染pmiκB的HeLa细胞，TNFα诱导了caspase8裂解活化及PARP裂解，而pBcl-xl／HA与pmiκB共转染、表达Bcl-xl的HeLa细胞，TNFα诱导的caspase8裂解活化及PARP裂解被阻断。结论在NFκB被抑制的实验细胞系统，Bcl-xl能阻断TNFα介导的细胞凋亡信号通路以及细胞凋亡，这对于进一步研究TNFα相关疾病发生机制及治疗措施有重要价值。

Bcl-xl blocks tumor necrosis factor alpha-induced caspase 8 activation and apoptosis

OBJECTIVE: To explore the effect of Bcl-xl on tumor necrosis factor-alpha (TNFalpha)-induced apoptosis signal pathway and apoptosis. METHODS: A dominant negative mutant of ikB (pmi kappaB) and Green Fluorescent Protein (GFP) expression plasmid pEGFP-C1, pmi kappab and pEGFP-C1 and Bcl-xl expression construct pBcl-xl/HA, were co-transfected into HeLa cells. Expression plasmid pBcl-xl/HA was introduced into p65-/-MEF cells in which nuclear factor-kappaB (NF-kappaB)/p65 was deficient, to establish cell line p65-/-Bcl-xl expressing Bcl-xl by selection with puromycin. These cells were treated with TNFalpha at a concentration of 10 ng/ml, and apoptotic cell death was examined microscopically with trypan blue staining. The proteins were abstracted from treated cells, and caspase 8 activation and cleavage of poly (ADP-ribose) polymerase (PARP) were examined by western blot using a specific antibody that recognized cleaved caspase 8 and cleaved PARP, respectively. RESULTS: HeLa cells transfected with pmi kappaB, TNFalpha showed significant cell death as they became rounded, shrank, and detached. However in HeLa cells co-transfected with pBcl-xl and pmi kappaB, no cell death was observed after treatment with TNFalpha. In p65-/- MEF cells; cell death was observed at 4 hours after treatment with TNFalpha, and cell death reached 90% at 12 hours after the treatment. However, in p65-/-Bcl-xl/HA cells expressing Bcl-xl, no cell death was seen even when treated with TNFa for 24 hours. Meanwhile, in pmikB/HeLa cells transfected with pmi kappaB, TNFalpha induced caspase 8 activation and PARP cleavage, but in the HeLa cells co-transfected with pBcl-xl and pmi kappaB, no activated caspase 8 and cleaved PARP were observed after treatment with TNFalpha. CONCLUSION: In the experimental system in which NF-kB was inhibited, Bcl-xl blocked TNFalpha-induced apoptosis signal pathway and apoptosis. These results bring to light that further studies of the pathogenesis and therapy of TNFa-related diseases are needed.