灭活仙台病毒Tianjin株诱导人乳腺癌细胞系BCAP37凋亡研究

Mechanism of apoptosis of breast cancer BCAP37 cells induced by inactivated Sendai virus Tianjin strain

目的:研究灭活仙台病毒Tianjin株诱导人乳腺癌细胞系BCAP37凋亡的作用。方法:用紫外线灭活不同浓度仙台病毒Tianjin株感染人乳腺癌BCAP37细胞,设立活病毒对照孔及正常细胞对照孔,感染后不同时间收集细胞,MTT法检测细胞存活率,琼脂糖凝胶电泳检测DNA片段降解情况,Hoechst染色观察细胞凋亡形态学变化,AnnexinⅤ/PI标记流式细胞仪检测细胞凋亡百分率,蛋白质印迹法检测Caspase-9及Caspase-3蛋白的表达。结果:MTT检测发现灭活仙台病毒Tianjin株对人乳腺癌细胞系BCAP37有明显的生长抑制作用,且呈剂量-时间依赖性;DNA片段琼脂糖凝胶电泳显示灭活病毒组呈典型的细胞凋亡梯形条带;Hoechst染色显示灭活病毒能够诱导BCAP37凋亡,显微镜下可见细胞核固缩,破裂,溶解,凋亡小体形成等形态学改变;流式细胞仪检测灭活病毒组细胞凋亡率为(15.1±1.1)%,明显高于正常细胞对照组,P<0.001;蛋白质印迹法结果显示,灭活病毒作用24h即出现Caspase-3及Caspase-9活化片段,与正常细胞组比较均明显升高,P值均<0.001。结论:灭活仙台病毒Tianjin株能够诱导人乳腺癌BCAP37细胞凋亡,其机制可能与Caspase-9和Caspase-3活化有关。

OBJECTIVE： To explore the apoptosis of breast cancer BCAP37 induced by inactivated Sendai virus Tian- jin strain. METHODS： The breast cancer BCAP37 cells were infected by different doses of UV-inactivated Sendal virus Tianjin strain and then collected at different time point, while live Sendal virus and negative controls were set up. The survival rate of cells was evaluated by MTT. The apoptosis of BCAP37 cell was detected by agrose gel electrophoresis of DNA fragments. Morphological change of apoptosis was examined by Hoechst fluorescent stain. The cell apoptosis rate was determined by Annexin V/PI staining and flow cytometry. Moreover, the expression levels of cleaved Caspase-9 and cleaved Caspase-3 were tested by western blot. RESULTS： MTT assay indicated that the survival rate of cells was dose- dependently decreased when treated with inactivated Sendai virus Tianjin strain. DNA gel electrophoresis demonstrated typical ladder of apoptosis in the inactivated Tianjin strain-treated group. Fluorescence staining with Hoechst 33258 showed nuclear condensation and apoptosis bodies in BCAP37 cell treated with inactivated Tianjin strain. Annexin V/PI staining and flow cytometry analysis showed that the apoptotic index was （15.1± 1.1） % in the inactivated Tianjin strain- treated group which was significantly higher than that in control group （P〈0. 001）. Western blot results indicated that inactivated Tianjin strain can improve the expressing of cleaved Caspase-3 and cleaved Caspase-9 in BCAP37 cell compared with control group at 24 h after infection （all P〈0. 001）. CONCLUSION： The inactivated Sendai virus Tianjin strain can induce the apoptosis in BCAP37 cells, which is associated with the activation the Caspase-3 and Caspase-9.