摘要
目的:研究3β,5α,6β-三羟基胆甾烷(Triol)诱导恶性胶质瘤细胞凋亡的作用及其机制。方法:以不同浓度的Triol作用于C6细胞和A172细胞不同时间。采用MTT法检测细胞存活率,Hoechst 33342染色和TUNEL法检测细胞凋亡,试剂盒检测caspase活性变化,蛋白免疫印记方法检测凋亡相关蛋白Bcl-2家族蛋白的变化。结果:Triol可呈剂量和时间依赖性降低C6细胞和A172细胞的存活率;Triol处理细胞48 h,C6细胞和A172细胞的IC50值分别为(17.8±0.6)μmol/L和(20.6±0.2)μmol/L。Hoechst 33342染色、TUNEL检测和凋亡执行酶caspase-3活性检测结果显示,给药组中2种细胞都出现明显凋亡核象、TUNEL阳性细胞数增多和caspase-3的激活。Triol作用于C6细胞12 h、24 h和48 h后,在凋亡外通路中激活的caspase-8和在凋亡内通路中激活的caspase-9活性均随时间升高,抗凋亡蛋白Bcl-2和Bcl-xL的表达量随时间降低,而促凋亡蛋白Bak的表达量随时间升高。结论:Triol通过激活内、外凋亡通路引起恶性胶质瘤细胞的凋亡,且Bcl-2家族蛋白在此过程中起重要的调控作用。
AIM： To investigate the effect of cholestane-3β, 5β, 6β-triol （Triol） on apoptosis of malignant glioma cells. METHODS： C6 cells and A172 cells were incubated with Triol at different concentrations for different time durations. MTT assay was used to detect the cell viability. Hoechst 3f3342 staining and TUNEL assay were used to analyze the cell apoptosis. The caspase activity was measured. The expression of apoptosis-related proteins, Bcl-2 family members, was determined by Western blotting. RESULTS： Triol decreased the cell viability of C6 and A172 cells in a dose- and time-dependent manner and the ICs0 values were （ 17.8 ＋0.6） Umol/L and （20.6 ~0.2） txmol/L, respectively. Visible nuclei with apoptotic characteristics, significant increase in TUNEL-positive cells, and the activation of apoptotic execution enzyme caspase-3 indicated that cell apoptosis was induced by Triol in both cell lines. After C6 cells were exposed to Triol for 12 h, 24 h and 48 h, the activity of caspase-8 in extrinsic apoptotic pathway and caspase-9 in intrinsic apoptotic path- way was increased time-dependently. Meanwhile, the levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL, was down-regula- ted, while pro-apoptotic protein Bak was up-regulated in a time-dependent manner. CONCLUSION： Triol induces apop- tosis of malignant glioma cells by activating intrinsic and extrinsic apoptotic pathways, and Bcl-2 family members are in- volved in Trial-induced apoptosis.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2013年第4期647-653,共7页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81173045)
广东华南新药创制中心神经与肿瘤药理学合作平台项目(No.2009ZX09301-015)
广东省自然科学基金资助项目(No.10451008901004893)
