羟基喜树碱抑制肺癌A549细胞的体外增殖并下调Bcl-2基因的表达

Hydroxycamptothecin inhibits proliferation of human lung carcinoma cell line A549 and down-regulates its Bcl-2 gene expression in vitro

目的研究羟基喜树碱对肺癌A549细胞生长抑制、细胞周期和细胞凋亡的影响。方法羟基喜树碱处理A549细胞后用吖啶橙/溴化乙锭(AO/EB)染色观察细胞形态学变化,琼脂糖凝胶检测DNA片段化,用流式细胞仪测定其对细胞周期影响。利用Annexin V-FITC/PI检测细胞凋亡,RT-PCR检测凋亡相关基因Bcl-2的表达变化。结果 A549细胞经羟基喜树碱作用,提取DNA进行琼脂糖凝胶电泳可见梯状条带。经AO/EB染色,荧光显微镜下观察到早期凋亡细胞细胞核被染成绿色,呈碎片状,晚期凋亡细胞核染色质为橘红色并呈固缩状或圆珠状,细胞周期S期由20%升高到60%。经1μmol/L羟基喜树碱处理24 h后,流式细胞术显示A549细胞的凋亡率为18.11%,明显高于对照组细胞凋亡率(0.09%,P<0.05)。经羟基喜树碱作用后,A549细胞Bcl-2基因表达下调70%(P<0.05)。结论羟基喜树碱可以明显抑制A549细胞增殖,诱导其凋亡,并下调Bcl-2基因表达,提示Bcl-2下调参与了羟基喜树碱对A549细胞的抑制作用。

Objective To investigate the effect of hydroxycamptothecin （HCPT） on the proliferation, cell cycle and apoptosis of human lung carcinoma cell line A549. Methods The growth of A549 cells exposed to HCPT was observed by staining with acridine orange/ethidium bromide dye. Agarose gel electrophoresis was performed to detect DNA fragmentation of the apoptotic cells. The cell cycle distribution of the exposed cells was analyzed using flow cytometry, and cell apoptosis was examined with annexin V-FITC/PI staining. RT-PCR was used to investigate Bcl-2 gene expression changes in the exposed cells. Results Agarose gel electrophoresis of the DNA from HCPT-treated cells showed a DNA ladder, and typical apoptotic appearance of the exposed cells was observed under fluorescence microscope. Treatment of A549 cells with 1 μmol/L HCPT for 24 h resulted in a cell apoptosis rate of 18.11%, significantly higher than the rate in control cells （0.09%, P〈0.05）. The treatment also caused a significant reduction of Bcl-2 mRNA expression by 70% （P〈0.05）. Conclusion HCPT can significantly inhibit the proliferation, induce apoptosis, and down-regulate Bcl-2 gene expression in human lung carcinoma cell line A549, suggesting the involvement of Bcl-2 gene in the inhibitory effect of HCPT on A549 cells.