人参皂苷Rh2诱导PC-3M细胞凋亡及其对新基因GRIM-19表达的影响

目的:研究人参单体皂苷Rh₂对激素非依赖性人前列腺癌细胞(PC-3M细胞株)的致凋亡作用及其对新基因GRIM-19表达的影响。方法:实验分为Rh₂高剂量组（40 mg·L^-1）、Rh₂中剂量组（30 mg·L^-1）、Rh₂低剂量组（15 mg·L^-1）及对照组（未加药组）。分别作用于PC-3M细胞24 h，吖啶橙染色观察其对PC-3M细胞的促凋亡作用；RT-PCR和Western blotting方法分别检测Rh₂对PC-3M细胞GRIM-19 mRNA及其蛋白表达的影响。结果:Rh₂高、中、低剂量组作用于PC-3M细胞24h吖啶橙染色均呈阳性结果。Rh₂低剂量组出现典型“出芽”现象，细胞中出现致密鲜亮橙色斑点。对照组细胞呈均匀绿色；RT-PCR结果显示，GRIM-19 mRNA随Rh₂浓度的增加表达逐渐增强，Rh₂中、低剂量组与对照组比较差异具有显著性（P<0.05），Rh₂高剂量组GRIM-19 mRNA表达与对照组比较差异具有高度显著性（P<0.01）。Western blotting结果与RT-PCR结果相一致，GRIM-19蛋白随Rh₂浓度的增加其表达逐渐增强，Rh₂中低剂量组与对照组比较差异具有显著性（P<0.05），Rh₂高剂量组GRIM-19蛋白的表达与对照组比较差异具有高度显著性（P<0.01）。结论:Rh₂诱导PC-3M细胞凋亡与上调GRIM-19基因和蛋白表达有关，其表达强度呈剂量依赖性。

Apoptosis of PC-3M cells induced by Rh2 and effects of Rh2 on expression of GRIM-19

Objective To study the effects of Rh_2(a ginsenoside extracted from ginseng) on PC-3M(androgen independent cell lines) apopotosis and new gene GRIM-19 expression.Methods Rh_2 agents were divided into three groups:high dose group(Rh_240 mg·L~(-1)),middle dose group(Rh_2 30 mg·L~(-1))and low dose group(Rh_2(15 mg·L~(-1)),)which incubated with PC-3M for 24 h, respectively,in the following experiments.Morphological assessment of apoptosis was performed by acridine orange(AO) stained fluorescence microscope to detect the apoptosis of PC-3M cells after treated with Rh_2 agents;RT-PCR was used to measure the level of GRIM-19 mRNA in PC-3M cells treated with Rh_2 agents;GRIM-19 protein expression of PC-3M cells treated with Rh_2 agent was measured by Western blotting.Results After various doses of Rh_2 interacted with PC-3M cells for 24 h,AO assay showed that cells changed from green color into orange one and typical exocytosis phenomena were observed in Rh_2(15 mg·L~(-1)) group.The consequences of RT-PCR and Western blotting demonstrated that GRIM-19 expression was intensified both in gene and protein levels with the increasing Rh_2 doses.The expressions of GRIM-19 in Rh_2(15 mg·L~(-1)) group and 30 mg·L~(-1) group were higher than that in control group(P<0.05),and in Rh_2(40 mg·L~(-1)) group the expression of GRIM-19 had a remarkable difference compared with control group(P<0.01).(Conclusion Rh_2 can) lead to apoptosis of PC-3M cells that correlated with GRIM-19 gene and protein expression upregulation,the expression level increases with the Rh_2 in a dose-dependent manner.