喜树碱诱导宫颈癌HeLa细胞凋亡及其机制

目的 测定不同浓度的喜树碱（CPT）对宫颈癌HeLa细胞促凋亡作用的影响，并探讨其促凋亡的机制。方法 人宫颈癌细胞系HeLa细胞，加入不同浓度的CPT（100 nmol/L~10 μmol/L），继续培养24 h。MTT方法检测细胞增殖，并通过倒置显微镜观察细胞形态学变化，细胞核染色法检测细胞凋亡，流式细胞仪检测细胞早期凋亡率。Western blotting法检测凋亡相关蛋白Bcl-2、Bax、cleave-Caspase-3、细胞色素C（Cyt-C）,聚腺苷二磷酸核糖聚合酶（PARP）的表达。结果 MTT实验及细胞形态学结果显示，CPT对HeLa细胞的生长有明显抑制作用，并可导致细胞形态改变，且具有剂量依赖性，其IC₅₀为2.45 μmol/L；细胞凋亡检测表明，不同浓度的CPT可导致凋亡小体出现，并可使早期凋亡比例增加。Western blotting法检测结果显示，不同浓度CPT可使HeLa细胞Bcl-2蛋白表达降低，而Bax、cleaved-Caspase-3蛋白表达，胞质Cyt-C，89 kD 5-磷酸核糖-1-焦磷酸（PRPP）蛋白表达增加且呈剂量依赖性。结论 CPT可以诱导HeLa细胞凋亡，其作用机制可能为激活线粒体依赖途径，并活化作用底物PARP而实现。

Effect of camptothecin on the proliferation and apoptosis of HeLa cells

Objective To study the effect of different concentrations of camptothecin(CPT) on the apoptosis of human cervical cancer cells (HeLa), and to explore the mechanism of apoptosis induced by CPT in HeLa cells. Methods The cultured in vitro human cervical cancer cells (HeLa) were divided into the control group and CPT groups treated with various concentrations(100 nmol/L-10 μmol/L). The viability of HeLa cells were detected by methyl thiazolyl tetrazolium (MTT) assay. The morphology changes of HeLa cells were observed with an inverted microscope. The cells apoptosis was analyzed by 4’6-diamidino-2-phenylindole(DAPI) and flow cytometry (FCM). Western blotting was used to detect the expression of Bcl-2, Bax,Cleave-Caspase-3，Cyt-C and poly ADP-ribose polymerase(PARP). Results Compared with the control group, the viability of HeLa cells decreased after treatment with different concentrations of CPT for 24 hours, and the cells morphology was changed in a dose-dependent manner. DAPI dying showed that there were apoptotic bodies FCM assay indicated that the early apoptosis ratios was increased after treatment with different concentrations of CPT for 24 hours. The cell apoptotic rate increased obviously in the CPT-treated groups than in the control group. The expression of Bcl-2 was decreased whereas the expressions of Bax,cleave-Caspase-3, Cyt-C into cell plasm and 89 kD phosphoribosyl pyrophosphate(PRPP) were increased when compared to that in the control group. Conclusion CPT can induce HeLa cell apoptosis, and its mechanism may be to activate the mitochondrial dependent pathway, and further activate substrate PARP.