论文部分内容阅读
目的探讨TAp63γ诱导的人食管鳞癌细胞株EC9706细胞凋亡的分子机制。方法用免疫组织化学和逆转录聚合酶链反应(RT-PCR)检测食鳞癌EC9706细胞中凋亡诱导因子(AIF)和p63的表达;将质粒pcDNA3.1-TAAp63γ转入EC9706细胞,流式细胞仪检测凋亡率,并用激光共聚焦显微镜和亚细胞器分离技术观察分析AIF的转化;采用RNA干扰技术下调AIF的蛋白表达水平,caspase广谱抑制剂zVAD.fmk预处理细胞,再用流式细胞仪分析EC9706细胞发达TAp63γ后调亡率的变化。结果EC9706细胞中AIF表达且定位在细胞质中,p63基因的表达亚型是ΔNp63,TAp63γ不表达;在pcDNA3.1-TAp63γ转染组和pcDNA3.1-p53转染组的细胞质及细胞核蛋白中分别检测到AIF,pc DNA3.1转染组的胞核蛋白无AIF信号;pcDNA3.1转染组、pcDNA3.1-TAp63γ转染组、AIF-siRNA干扰后再转染pcDNA3.1-TAp63γ组、zVAD.fmk处理后再转染pcDNA3.1-TAp63γ组和AIF-siRNA干扰联合zVAD.fmk处理后再转染pcDNA3.1-TAp63γ的凋亡率分别为1.37%、13.64%、4.52%、4.03%和1.91%。结论TAp63γ在诱导EC9706细胞凋亡的过程中,AIF从线粒体释放入胞质并转位到细胞核内;下调AIF的蛋白表达水平可降低TAp63γ诱导的细胞凋亡;TAp63γ诱导的细胞凋亡依赖于AIF和caspase。
Objective To investigate the molecular mechanism of TAp63γ-induced esophageal squamous cell carcinoma cell line EC9706 apoptosis. Methods The expression of apoptosis-inducing factor (AIF) and p63 in esophageal squamous carcinoma cell line EC9706 were detected by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The pcDNA3.1-TAAp63γ plasmid was transfected into EC9706 cells by flow cytometry AIF was detected by laser scanning confocal microscopy and subcellular organelle separation. The expression of AIF protein was down-regulated by RNAi and pretreated by caspase-inhibitor zVAD.fmk. Flow cytometry Cytometry analysis of EC9706 cells after the development of TAp63γ changes in the rate of death. Results The expression of AIF in EC9706 cells was localized in the cytoplasm. The subtypes of p63 gene were ΔNp63 and not expressed in TAp63γ. In the cytoplasm and nucleoprotein of pcDNA3.1-TAp63γ transfected and pcDNA3.1-p53 transfected groups, The AIF was detected in the nuclei of pcDNA3.1 transfected group; pcDNA3.1 transfected group, pcDNA3.1-TAp63γ transfected group, AIF-siRNA transfected pcDNA3.1-TAp63γ group, The apoptotic rates of pcDNA3.1-TAp63γ transfected with pcDNA3.1-TAp63γ and zIF-siRNA after zVAD.fmk treatment combined with zVAD.fmk were 1.37%, 13.64%, 4.52%, 4.03% And 1.91%. Conclusions TAp63γ is released from mitochondria into the cytoplasm and translocates into the nucleus during the induction of apoptosis in EC9706 cells. Down-regulation of AIF protein expression can reduce the apoptosis induced by TAp63γ. TAp63γ-induced apoptosis is dependent on AIF And caspase.