益气养阴除结方联合顺铂对肺腺癌A549细胞生长及凋亡的影响

Effects of Yiqi Yangyin Chujie Formula combined with cisplatin on the growth and apoptosis of human lung adenocarcinoma cell A549

目的探讨益气养阴除结方联合顺铂对肺腺癌细胞（A549细胞）凋亡的影响及其机制。方法选用A549细胞培养,实验分为4组,分别为空白对照组、益气养阴除结方组（简称中药组）、顺铂组、联合组。空白对照组不加药物处理;中药组浓度梯度设定分别为1 000、500、200、100、50、20 mg/L;顺铂组浓度梯度设定分别为4 mg/L和8 mg/L;联合组浓度梯度分别为以上2组之间的组合。应用MTT法检测药物对其增殖的抑制作用;TUNNEL法检测细胞凋亡情况;免疫细胞化学法检测肿瘤细胞Bcl-2及Caspase-3蛋白的表达。结果与空白对照组比较,中药组、顺铂组及联合组的细胞抑制率均有明显增加（P〈0.05~P〈0.001）,其中联合组（中药200 mg/L＋顺铂4mg/L）抑制率最高;各用药组凋亡细胞数量均有明显增加（P〈0.01,P〈0.001）,其中联合组（中药200 mg/L＋顺铂4 mg/L）凋亡细胞数量最多;各用药组bcl-2的表达均降低,其中顺铂组（4 mg/L）、联合组（中药200 mg/L＋顺铂4 mg/L）Bcl-2的表达明显降低（P〈0.01）;各用药组Caspase-3表达的表达均升高,其中中药组（200 mg/L）、联合组（中药200 mg/L＋顺铂4 mg/L）有显著性差异（P〈0.05,P〈0.01）。结论该中药复方能促进A549细胞凋亡抑制肿瘤细胞生长,联合一定浓度顺铂后其效果更明显,其机制可能与抑制Bcl-2和增加Caspase-3的表达有关。

Objective To investigate the effects of Yiqi Yangyin Chujie Formula（ qi-tonifying yin-nourishing mass-eliminating formula） combined with cisplatin on the apoptosis of human lung adenocarcinoma cell A549. Methods Human lung adenocarcinoma cell line A549 was cultured in vitro and then divided into four groups： blank control group,Yiqi Yangyin Chujie Formula group（ TCM group）,cisplatin group and combined treatment group. The blank control group received no intervention. TCM group was treated with Yiqi Yangyin Chujie Formula at doses of 1 000 mg / L,500 mg / L,200 mg / L,100 mg / L,50 mg / L and 20 mg / L respectively. Cisplatin doses were set at 4 mg / L or 8 mg / L while the combined treatment group was treated with both cisplatin and TCM formula at dose combinations mentioned above. Then cell proliferation inhibition was tested by using tetrazolium（ MTT） colorimetric assay. Terminally labeled transferase technique（ TUNNEL） apoptosis of A549 cells was applied. Expression of Bcl-2 and Caspase-3 were determined by using immunocytochemistry technique. Results MTT assay showed that the inhibition rates of experiment group were significantly higher than those of control group（ P〈0. 05 ~ P 0. 001） with the combined group（ 200 mg / L TCM ＋ 4 mg / L cisplatin） topping the list. TUNNEL showed that numbers of apoptotic cells in the experiment groups were significantly higher than that of the control group（ P〈0. 01 ~ P 0. 001） with the highest number observed in the combined treatment group（ 200 mg / L TCM ＋ 4 mg / L cisplatin）. Expression of Bcl-2 decreased in all experiment groups compared with that of control group. The reduction was significant（ P〈0. 01） in cisplatin group（ 4 mg /L） and combined treatment group（ 200 mg / L TCM ＋ 4 mg / L cisplatin）. Expression of Caspase-3 increased in all experiment groups compared with that of control group. The increase（ P〈0. 05,P 0.01） was significant in TCM group（ 200 mg / L） and combined treatment group（ 200 mg / L TCM ＋ 4 mg /L cisplatin）. Conclusion Yiqi Yangyin Chujie Formula could inhibit the proliferation of A549 cell through inducing apoptosis of A549,which appears to be more effective when combined with cisplatin.Its mechanism may be related with the suppression of Bcl-2 and enhancement of Caspase-3 expression.