土槿皮乙酸诱导大鼠神经胶质瘤C6细胞株凋亡

摘要

目的:研究土槿皮乙酸诱导大鼠神经胶质瘤C6细胞株凋亡及其机制.方法:采用MTT法检测0、0.625、1.25、2.5、5、10、20、40、80 μmol/L不同终浓度PAB对C6细胞增殖能力的影响,激光扫描共聚焦显微镜观察AO/EB荧光染色后细胞的凋亡情况,流式细胞术检测碘化丙啶(PI)染色后细胞周期的变化,免疫组化法测定C6细胞的凋亡相关蛋白Bcl-2的表达.结果:MTT结果显示,PAB呈时间剂量依赖性抑制C6细胞的生长.PAB处理24、48和72 h后,对C6细胞的半效致死剂量(IC50)分别为39.811、23.306和7.360 μmol/L.5.0、10.0、20.0 μmol/L PAB作用C6细胞72 h后的凋亡率分别为(50.5±0.7)%、(63.5±2.3)%和(80.0±1.2)%,与对照组(12.0±1.6)%比较均明显升高,差异有统计学意义(P<0.05).流式细胞仪检测结果显示5.0、10.0、20.0 μmol/L不同浓度PAB作用C6细胞72小时后,随着浓度的增加,G2/M期细胞逐渐增多,分别为(29.84±6.215)%,(34.58±1.187)%和(70.23±6.325)%,与对照组(7.79±0.570)%相比,后两组差异有统计学意义(P<0.05).C6细胞中Bcl-2蛋白阳性表达率随药物剂量的增大而减小.结论:PAB可抑制大鼠神经胶质瘤C6细胞增殖,阻滞细胞周期于G2/M期、下调Bcl-2表达为其可能的作用机制.%Objective:To investigate the action mechanisms for the effect of pseudolaric acid B in inducing apoptosis of murine C6 glioma cell line.Methods:The effects of different mass concentrations of PAB (0,0.625,1.25,2.5,5,10,20,40,80 μmol/L) on the growth of C6 cells were analysed by MTT assay.The apoptosis effect of C6 cells was detected by inverted UV fluoresence microscope with AO/EB staining.Cell cycle was checked by flow cytometry with PI staining.The expression of Bcl-2 protein (apoptosis associated protein) was determined by immunohisto-chemistry.Results:MTT assay revealed PAB inhibited the growth of C6 cells in a dose and time dependent manner.The median lethal dosages (IC50) of PAB on C6 cells were 39.811,23.306 and 7.360 μmol/L after treatment with PAB for 24,48 and 72 h respectively.The apoptosis rates were (50.5±0.7)%,(63.5±2.3)% and (80.0±1.2)% after treatment with PAB (5.0,10.0,20.0 μmol/L) for 72 h respectively,they were all significantly different from that of control group (12.0±1.6)%(P<0.05).Flow cytometry indicated that the increasing of C6 cells in G2/M phase occurred 72 h after treatment with increasing of the concentration of PAB (5.0,10.0 and 20.0 μmol/L),and the rates were (29.84±6.215)%,(34.58±1.187)% and (70.23±6.325)% respectively,the last two were significantly different from that of control group (7.79±0.570)% (P<0.05).The expression of Bcl-2 protein in C6 cells decreased with the increasing of concentration of PAB.Conclusion:PAB can inhibit the growth of murine glioma C6 cells and induce cell apoptosis,which may be associated with the decreased expression of Bcl-2 and arrested in G2/M phase.