沉默Bax基因对TNF-α诱导人肺泡Ⅱ型上皮细胞凋亡的影响

siRNA knocks down Bax gene and inhibits TNF-α induced apoptosis in A549 cells

目的:运用RNA干扰(RNAi)技术沉默人肺泡Ⅱ型上皮细胞株A549的B细胞淋巴瘤-2相关基因(Bax),探讨Bax在肿瘤坏死因子-α(TNF-α)诱导A549细胞凋亡中的作用。方法:体外培养A549细胞,利用脂质体LipofectamineTM2000将化学合成的Bax小干扰RNA(siRNA)转染入A549细胞,给予10μg/LTNF-α刺激24h,RT-PCR检测bax mRNA的表达,Western blotting和免疫组化检测Bax蛋白的表达,流式细胞术检测细胞凋亡率的变化。结果:化学合成的Bax siRNA抑制了A549细胞Bax mRNA和蛋白的表达(P<0.05),流式细胞术显示BaxsiRNA组的细胞凋亡率明显低于TNF-α组和阴性对照siRNA组(P<0.05)。结论:体外实验证明Bax的高表达在TNF-α诱导A549细胞凋亡中发挥了重要的促凋亡作用,利用RNAi技术沉默Bax基因可以有效抑制由TNF-α介导的A549细胞凋亡。

AIM：To investigate the influence of tumor necrosis factor-α （TNF-α） induced apoptosis through knocking down Bax gene by RNA interference （RNAi） in human alveolar type II epithelial cells （A549）. METHODS： A549 cells were cultured in vitro and divided into 4 groups according to RNAi treatment： control group,TNF-α treated group,Bax siRNA group and control siRNA group. Chemically synthesized small interfering RNA （siRNA） directed against human Bax gene was transfected into A549 cells by cationic liposome. The effect of RNAi was investigated by reverse transcription PCR,Western blotting and immunohistochemisty,and the rate of apoptosis was investigated by flow cytometry. RESULTS： Bax gene was knocked down effectively in Bax siRNA group （P〈0.05）. Apoptosis was induced by TNF-α in TNF-α treated group and control siRNA group. However,it was abolished in Bax siRNA group by the downregulation of Bax gene compared to TNF-α treated group and control siRNA group （P〈0.05）. CONCLUSION： This result suggests that Bax gene has a significant role of pro-apoptosis in A549 cells and knocking down Bax gene by RNAi can effectively inhibit TNF-α-induced apoptosis in A549 cells.