论文部分内容阅读
目的阐明iASPP在p53缺失细胞中影响细胞凋亡的机制。方法将iASPP、干扰iASPP、p63、p73、Bax-Luc和Puma-Luc等转染进H1299细胞中,观察各种情况下对前凋亡基因转录能力的影响;用体外转录/翻译偶联系统、免疫沉淀和Western blot方法研究iASPP与p63和p73在体外的相互作用;用细胞转染、免疫沉淀和Western blot方法研究iASPP与p63和p73在细胞内的相互作用。结果①H1299细胞转入iASPP后前凋亡基因Bax和Puma的转录能力下降,分别只有对照组的0.14倍和0.13倍;②H1299细胞转入iASPP干扰质粒后前凋亡基因Bax和Puma的转录能力增强,分别为对照组的3.58倍和4.14倍;③p63和p73转染H1299细胞后,前凋亡基因Bax和Puma启动子的转录能力增强,分别增加4.67倍和3.03倍(转染p63后)和5.90倍和3.36倍(转染p73后);④iASPP与p63、p73在体外和细胞内存在着相互作用;⑤干扰iASPP后p63和p73的蛋白表达水平不变。结论在p53缺失的H1299细胞中,iASPP是通过抑制p63/p73的转录因子的功能,从而影响前凋亡基因Bax、Puma等的转录活力,进而导致细胞凋亡变化的。
Objective To elucidate the mechanism of iASPP affecting apoptosis in p53-deficient cells. Methods The iASPP, iASPP, p63, p73, Bax-Luc and Puma-Luc were transfected into H1299 cells to observe the effect of iASPP on the transcription of proapoptotic genes. Using in vitro transcription / translation coupling system, The interaction of iASPP with p63 and p73 in vitro was studied by immunoprecipitation and Western blot. The interaction between iASPP and p63 and p73 in cells was studied by cell transfection, immunoprecipitation and Western blot. Results ① The transcriptional abilities of Bax and Puma were decreased after H1299 cells transfered into iASPP, which were only 0.14 times and 0.13 times that of the control group respectively. ② The transcriptional capacity of Bax and Puma was increased after H1299 cells were transfected with iASPP, Which was 3.58-fold and 4.14-fold higher than that of the control group, respectively. (3) After transfected with p63 and p73, the transcriptional capacity of Bax and Puma promoters increased 4.67 and 3.03 fold (after p63 transfection) and 5.90 fold And 3.36 folds (after transfection with p73); ④iASPP interacted with p63 and p73 in vitro and in vivo; ⑤ The protein expression levels of p63 and p73 did not change after interference with iASPP. Conclusions In p53-deficient H1299 cells, iASPP affects the transcriptional activity of pro-apoptotic genes Bax, Puma and so on by inhibiting the function of p63 / p73 transcription factor, leading to changes of apoptosis.