沉默Bax基因对TNF-α诱导肺泡上皮细胞凋亡的影响

Effects of silencing Bax gene on apoptosis of alveolar epithelial cells induced by TNF-α

目的探讨沉默Bcl-2相关X蛋白(Bax)对肿瘤坏死因子-α(TNF-α)诱导肺泡上皮细胞凋亡的影响。方法用0 ng/ml、10 ng/ml的TNF-α作用于人A549细胞,RT-PCR和Western blot检测细胞中Bax表达水平。A549细胞转染Bax小干扰RNA(Bax siRNA)、小干扰RNA阴性对照(siRNA control),RT-PCR和Western blot检测细胞中Bax表达水平。细胞分为空白对照组(未转染细胞)、TNF-α组(未转染细胞,培养液中含有10 ng/ml的TNF-α)、阴性对照组(转染siRNA control后的细胞,培养液中含有10 ng/ml的TNF-α)、干扰组(转染Bax siRNA后的细胞,培养液中含有10 ng/ml的TNF-α)。流式细胞术检测各组细胞凋亡情况,Western blot检测细胞中p38、磷酸化的p38(p-p38)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)的表达。结果 10 ng/ml的TNF-α作用后的细胞中Bax mRNA和蛋白表达水平升高。转染Bax siRNA后细胞中Bax mRNA和蛋白水平均降低。空白对照组、TNF-α组、阴性对照组、干扰组细胞凋亡率依次为:(0.92±0.01)%、(7.94±0.19)%、(7.93±0.17)%、(4.62±0.13)%。TNF-α组、阴性对照组、干扰组细胞中p-p38、Cleaved Caspase-3表达水平均明显高于空白对照组(P<0.01)。干扰组细胞中p-p38、Cleaved Caspase-3表达水平均明显低于TNF-α组(P<0.01)。结论 TNF-α能够诱导肺泡上皮细胞凋亡,而沉默Bax能够部分逆转TNF-α促凋亡作用,作用机制可能与p38信号通路有关。

Objective To investigate the effects of silencing Bax on apoptosis of alveolar epithelial cells induced by TNF-α. Methods TNF-α of 0 ng/ml,10 ng/ml was applied on human A549 cells. The levels of Bax was detected by RT-PCR and Western blot. A549 cells transfected with Bax small interfering RNA（ Bax siRNA）,small interfering RNA negative control（ siRNA control）. The Bax expression in cells was detected by RT-PCR and Western blot. Cells were divided into blank control group（ non transfected cells）,TNF-α group（ non transfected cells,culture medium containing 10 ng/ml of TNF-α）,negative control group（ transfected siRNA control cells,culture medium containing 10 ng/ml of TNF-α）,interference group（ transfected Bax siRNA cells,culture medium containing 10 ng/ml of TNF-α）. Cell apoptosis was detected by flow cytometry,the expression of p38,p-p38 and Cleaved in Caspase-3 cells was detected by Western blot. Results The levels of Bax mRNA and protein expression in cells elevated with 10 ng/m L TNF-α. The levels of Bax mRNA and protein decreased after transfection with Bax siRNA. Blank control group,TNF-α group,negative control group,interference group apoptosis rate were（ 0. 92 ± 0. 01） %,（ 7. 94 ±0. 19） %,（ 7. 93 ± 0. 17） %,（ 4. 62 ± 0. 13） %. The expression levels of p-p38 and Cleaved Caspase-3 in TNF-α group,negative control group and interference group were significantly higher than those in the blank control group（ P 〈 0. 01）. The expression levels of p-p38 and cleaved caspase-3 in the interference group were significantly lower than those in TNF-α group（ P 〈 0. 01）. Conclusion TNF-α can induce the apoptosis of alveolar epithelial cells,while Bax can partly reverse the apoptosis promoting effect of TNF-α,which may be related to p38 signaling pathway.