黄芪诱导人肺癌A549细胞凋亡及作用机制的初步研究

Study on Apoptosis-inducing Effect of Astragalus and the Mechanism on Human Lung Cancer A549 Cell

目的探讨黄芪对人非小细胞肺癌A549细胞增殖和凋亡的影响及促进A549细胞凋亡的分子机制。方法体外培养并取处于对数生长期的A549细胞用于实验,分别给予不同浓度的黄芪注射液(0.1 g/ml、0.5 g/ml、1.0 g/ml、1.6 g/ml)进行干预,作用24 h和48 h后采用CCK-8法测定吸光度值并计算细胞增殖抑制率。流式细胞术(FCM)检测不同浓度的黄芪注射液干预24 h和48 h后的细胞凋亡情况。采用逆转录-聚合酶链式反应(RT-PCR)法检测各组细胞中凋亡相关基因Survivin mRNA、Bcl-2 mRNA及Bax mRNA的表达。免疫印迹法(Western blot)检测各组细胞中凋亡相关蛋白Survivin、Bcl-2及Bax表达的情况。结果与空白对照组相比较,经黄芪注射液干预后能够显著提高人非小细胞肺癌A549细胞的增殖抑制率,且黄芪注射液该作用具有一定的时间依赖性和浓度依赖性。流式细胞术检测细胞凋亡分析表明:随药物浓度的增高、药物作用时间的延长,早期凋亡和晚期凋亡细胞的比例都逐渐增加,且与空白对照组比较具有显著差异。黄芪注射液能下调凋亡抑制基因Survivin mRNA、Bcl-2 mRNA的表达,上调促凋亡基因Bax mRNA的表达。黄芪注射液能下调凋亡抑制蛋白Survivin、Bcl-2的表达,上调促凋亡蛋白Bax的表达。结论黄芪注射液能够有效的抑制人非小细胞肺癌A549细胞增殖并呈现出时间和浓度依赖性,这种抑制作用与药物调节凋亡相关基因和蛋白质表达有关。

Objective To investigate the effects of Astragalus on the proliferation and apoptosis of human non-small cell lung cancer(NSCLC)A549 cells and the molecular mechanism of promoting the apoptosis of A549 cells.Methods A549 cells in logarithmic phase were cultured in vitro and interfered with different concentrations of Astragalus Injection(0.1 g/ml,0.5 g/ml,1.0 g/ml,1.6 g/ml).After 24 hours and 48 hours,the absorbance was measured by CCK-8 method and the inhibition rate of cell proliferation was calculated.Flow cytometry(FCM)was used to detect the apoptosis of cells treated with different concentrations of Astragalus Injection for 24 hours and 48 hours.The expression of apoptosis-related genes Survivin mRNA,Bcl-2 mRNA and Bax mRNA were detected by reverse transcription-polymerase chain reaction(RT-PCR).Western blotting was used to detect the expression of apoptosis related proteins Survivin,Bcl-2 and Bax in each group.Results Compared with the blank control group,Astragalus Injection could significantly improve the proliferation inhibition rate of human non-small cell lung cancer A549 cells,and the effect of Astragalus Injection was time-dependent and concentration-dependent.The analysis of apoptosis detected by flow cytometry showed that with the increase of drug concentration and the extension of drug action time,the proportion of early and late apoptotic cells gradually increased,and there was significant difference compared with the blank control group.Astragalus Injection can down-regulate the expression of apoptosis-inhibiting gene Survivin mRNA and Bcl-2 mRNA,and up-regulate the expression of apoptosis-promoting gene Bax mRNA.Astragalus Injection can down-regulate the expression of apoptosis-inhibiting protein Survivin and Bcl-2,and up-regulate the expression of apoptosis-promoting protein Bax.Conclusion Astragalus Injection can effectively inhibit the proliferation of human non-small cell lung cancer A549 cells in a time-and concentration-dependent manner,which is related to the drug regulation of apoptosis-related gene and protein expression.