Leydig肿瘤细胞中GR表达量改变与皮质酮诱导的细胞凋亡的相关性

Correlation of changes of GR expression in Leydig tumor cells and cell apoptosis induced by corticosterone

目的以小鼠Leydig肿瘤细胞(MLTC)为细胞模型,探讨细胞中糖皮质激素受体(GR)表达量改变与应激水平皮质酮(CORT)诱导的细胞凋亡的相关性。方法 GR过表达实验分为GR质粒组、空载质粒组和空白对照1组,采用基因克隆技术使GR质粒组MLTC中的GR呈过表达状态,Western blotting检测验证;GR低表达实验分为空白对照2组、脂质体对照组、GRsiRNA组和对照siRNA组,采用RNA干扰技术使GR siRNA组MLTC中的GR呈低表达状态,Western blotting检测验证。以应激水平的CORT(终浓度200 nmol/L)处理两种实验分组的MLTC(GR过表达+CORT组、空载质粒+CORT组、空白对照1组和GR低表达+CORT组、对照siRNA+CORT组、空白对照2组),分别以CORT处理的空白对照1、2组MLTC作为单纯CORT处理1、2组;流式细胞术检测各组MLTC细胞凋亡率,分析GR表达与细胞凋亡率的相关性。结果 GR过表达+CORT组、空载质粒+CORT组、单纯CORT处理1组和空白对照1组的细胞凋亡率分别为(36.6±0.6)%、(23.8±0.8)%、(24.3±0.6)%和(4.1±0.5)%,GR过表达+CORT组细胞凋亡率显著高于其他各组(P<0.01);GR低表达+CORT组、对照siRNA+CORT组、单纯CORT处理2组和空白对照2组的细胞凋亡率分别为(12.2±0.6)%、(22.5±0.6)%、(22.3±0.9)%和(4.1±0.5)%,;GR低表达+CORT组细胞凋亡率显著低于对照siRNA+CORT组和单纯CORT处理2组(P<0.01)。MLTC中GR表达量与细胞凋亡率呈显著正相关(r=0.947,P<0.05)。结论 Leydig细胞中GR表达量的改变与应激水平CORT诱导的细胞凋亡率改变呈显著正相关,由应激导致的Leydig细胞凋亡可能是糖皮质激素直接作用的结果。

Objective To investigate the correlation of glucocorticoid receptor(GR) expression in mouse Leydig tumor cells(MLTC) and cell apoptosis induced by corticosterone(CORT) at stress level. Methods In the experiment with GR over-expression,blank control group 1,GR plasmid group and control plasmid group were established,gene cloning was used to achieve the over-expression of GR in MLTC in GR plasmid group,and identification was performed by Western blotting.In the experiment with knockdown of GR expression,blank control group 2,Lipofectamine control group,GR siRNA group and control siRNA group were set,RNA interference was adopted to achieve the knockdown of GR in MLTC,and identification was performed by Western blotting.MLTC in these two experiments were treated by CORT at stress level(200 nmol/L)(GR over-expression+CORT group,control plasmid+CORT group,blank control group 1,and knockdown of GR expression+CORT group,control siRNA+CORT group and blank control group 2),MLTC in blank control group 1 and 2 treated by CORT were served as single CORT treatment group 1 and 2,apoptotic rate of MLTC in each group was detected by flow cytometry,and correlation of GR expression and cell apoptotic rate was analysed. Results The cell apoptotic rates in GR over-expression+CORT group,control plasmid+CORT group,single CORT treatment group 1 and blank control group 1 were(36.6±0.6)%,(23.8±0.8)%,(24.3±0.6)% and(4.1±0.5)% respectively,and the cell apoptotic rate of GR over-expression+CORT group was significantly higher than the other groups(P<0.01).The cell apoptotic rates in knockdown of GR expression+CORT group,control siRNA+CORT group,single CORT treatment group 2 and blank control group 2 were(12.2±0.6)%,(22.5±0.6)%,(22.3±0.9)% and(4.1±0.5)% respectively,and the cell apoptotic rate of knockdown of GR expression+CORT group was significantly lower than control siRNA+CORT group and single CORT treatment group 2(P<0.01).The expression of GR in MLTC was significantly positively related to cell apoptotic rate(r=0.947,P<0.05). Conclusion The changes of GR expression in Leydig cells are significantly positively related to the changes of cell apoptotic rates induced by CORT at stress level,and the glucocorticoid has direct effect on Leydig cell apoptosis induced by stress.