雷公藤甲素诱导肝细胞L-02凋亡的作用机制研究

摘要

目的:研究雷公藤甲素诱导肝细胞L-02凋亡的作用机制.方法:体外培养L-02细胞,MTT法检测雷公藤甲素对L-02细胞的毒性作用,Hoechst 33258染色观察凋亡细胞核形态的改变,流式细胞仪检测细胞凋亡率,Western blot法检测人抗兔p53、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)与线粒体、胞浆中细胞色素C(Cyt-c)的表达.结果:不同浓度雷公藤甲素(60、120、180 nmol/L)与同样浓度(120 nmol/L)不同作用时间(12、24、48 h)作用L-02细胞,雷公藤甲素对细胞的抑制作用与浓度或时间呈正相关；Hoechst 33258染色可见细胞出现典型的凋亡细胞形态学改变；Western blot法显示120 nmol/L雷公藤甲素作用后,细胞p53 、Bax与胞浆中Cyt-c表达显著增强(P＜0.05),而Bcl-2与线粒体中Cyt-c表达显著减弱(P＜0.05).结论:雷公藤甲素体外可诱导L-02细胞凋亡,其作用机制可能与Bcl-2与线粒体中Cyt-c表达下调和p53、Bax与胞浆中Cyt-c表达上调有关.%OBJECTIVE: To study the mechanism of triptolide-induced L-02 cells apoptosis. METHODS: L-02 cells were cultured in vitro. The inhibitory effect of triptolide on the proliferation of L-02 cells was evaluated using MTT assay. Hoechst 33258 fluorescent staining was used to observe themorphological changes of apoptotic cells. Flow cytometry was performed to determine the apoptotic rate. The protein expression of p53, Bcl-2, Bax, Cyt-c in mitochondria and cytosol were detected using Western blot analysis. RESULTS: L-02 cells are treated with different concentrations of triptolide (60, 120 and 180 nmol/L) and same concentration of triptolide for different times (12, 24 and 48 h). It showed that inhibition effect of triptolide on cell proliferation was positively correlated with the concentration of triptolide or the time. Hoechst 33258 staining revealed typical apoptotic morphological changes. Western blot analysis suggested that the contents of p53, Bax and cytosol Cyt-c enhanced significantly (P<0.05) , and Bcl-2 and the content of Cyt-c in mitochondria weakened significantly (P<0.05) after treatment with triptolide 120 nmol/L. CONCLUSION: Triptolide probably may induce the apoptosis of L-02 cells by up-regulating proteins expression of Cyt-c in p53, Bax and cytosol and down-regulating proteins expression of Cyt-c in Bcl-2 and mitochondria.