冬凌草甲素诱导肝癌细胞凋亡中Bcl-2及端粒酶变化的研究

Change of Bcl-2 expression and telomerase during apoptosis induced by oridonin on human hepatocelluar carcinoma cells

目的:探讨冬凌草甲素诱导人肝癌细胞BEL-7402凋亡中Bc l-2表达及端粒酶活性的变化。方法:以8,16,24,32μmol.L-1不同浓度的冬凌草甲素作用于体外培养的BEL-7402细胞,应用流式细胞仪检测细胞凋亡,Hoechst 33258荧光染色法。W estern b lot观察Bc l-2,Bax表达变化。应用PCR-ELISA及RT-PCR法检测细胞凋亡前后端粒酶活性及端粒酶hTERT mRNA表达水平的变化。结果:冬凌草甲素可诱导肝癌细胞BEL-7402发生细胞凋亡,具有明显的量-效与时-效关系。冬凌草甲素作用60 h后,在Hoechst 33258荧光染色图片上可见核浓缩及核碎裂等典型的细胞凋亡特征。凋亡过程中Bc l-2蛋白表达的降低和Bax蛋白上调,同时端粒酶hTERT mRNA的表达水平及端粒酶活性均明显降低。结论:冬凌草甲素在诱导肝癌细胞BEL-7402发生细胞凋亡过程中,能降低端粒酶hTERT mRNA的表达水平和端粒酶活性,同时抗凋亡蛋白Bc l-2表达的降低和促凋亡蛋白Bax上调。

Objective： To investigate the change of Bel-2 expression and telomerase during the apoptosis induced by oridonin on human hepatocelluar carcinoma BEL7402 cells. Method： BEL-7402 cells in culture medium were given 8,16,24,32μmol · L^-1 different concentrations of oridonin. The cell apoptotic rate was detected by flow cytometry（ FCM）, morphology of cell apoptosis was observed by Hoeehst 3325 staining. Bcl-2 and Bax expressions were detected by Western blotting. Reverse transcriptase polymerase chain reaction（RT-PCR） and PCR enzyme-linked immunosorbent assay （EIJSA） were used to detect hTERT mRNA expression and telomerase activity. Result： Oridonin induced BEL-7402 cells apoptosis significantly, and the apoptosis rate was both in time-and dose-dependent manner. Marked morphological changes of cell apoptosis were observed very clearly by Hoechst 33258 staining after the cells exposed to oridonin for 60 hours; Western blotting showed that Bcl-2 expression was down-regulated and Bax expression up-regulated concurrently along with the apoptotic process, and the expression of hTERT mRNA as well as activity of telomerase were decreased con- currently. Conclusion ： Oridonin could decrease the expression of hTERT mRNA and telomerase activity as well as down-regulation of Bcl-2 and up-regulation of Bax expression during the apoptosis of BEL-7402 cells.