膜联蛋白5对大鼠睾丸间质细胞增殖过程中Ect2表达的影响

目的研究膜联蛋白5(annexin 5)对大鼠睾丸间质细胞增殖过程中上皮细胞转化序列2癌基因(epithelial cell transfor-ming sequence 2 oncogene,Ect2)表达的影响,探讨annexin 5影响睾丸间质细胞增殖的机制。方法原代培养大鼠睾丸间质细胞,不同剂量的annexin 5处理后,采用MTT法检测细胞增殖活力,流式细胞术检测细胞周期,RT-PCR检测Ect2的mRNA表达改变,western blot分析睾丸间质细胞中Ect2蛋白质表达变化。结果 MTT法检测结果显示,annexin 5对大鼠睾丸间质细胞增殖有明显的促进作用,且在2～3 d呈显著的剂量-时间依赖关系(P<0.01),在第5天细胞增殖作用已明显减弱。流式细胞分析发现,1 nmol/L annexin 5作用48 h时G2/M期细胞减少为24.49%,72 h时减少为16.43%,与对照组比较差异显著(P<0.05)。RT-PCR结果表明,0.1 nmol/L组和1 nmol/L组Ect2 mRNA表达[(0.77±0.06)和(0.85±0.04)]与对照组(0.67±0.06)比较,分别增加了14.9%(P<0.05)和26.9%(P<0.01),而10 nmol/L组Ect2 mRNA表达与对照组比较差异无统计学意义(P>0.05)。western blot结果表明,1 nmol/L annexin 5作用组的Ect2蛋白质表达比对照组增加了20.9%[(1.50±0.15)vs(1.24±0.07),P<0.05],而0.1 nmol/L组和10 nmol/L组与对照组比较差异无统计学意义(P>0.05)。结论 annexin 5对大鼠睾丸间质细胞增殖的促进作用是通过促进细胞周期由G2期向M期的转变来实现的。Ect2在基因和蛋白质水平均受annexin 5的影响,提示annexin 5调控睾丸间质细胞增殖可能通过增加Ect2的表达而实现。

Effects of annexin 5 on expression of Ect2 in rat Leydig cells during proliferation process

Objective To research the effects of annexin 5 on the expression of epithelial cell transforming sequence 2 oncogene(Ect2) in rat Leydig cells,and discuss the mechanism of cell proliferation affected by annexin 5.Methods The primary Leydig cells from rats were cultured in vitro and treated with different concentration of annexin 5.The proliferation rates of Leydig cells were measured by MTT assay.Phase distributions of cell cycle were analyzed by flow cytometry.The levels of mRNA and protein of Ect2 were determined by RT-PCR and western blot respectively.Results MTT assay showed that annexin 5 promoted the growth of rat Leydig cells in a time-dependent manner for the first 2 to 3 days.The proliferation of Leydig cells significantly reduced on the 5th day.Flow cytometry analysis revealed that the proportion of cells inG2/M phase decreased from 28.39 % to 24.49% following treatment with 1 nmol/L of annexin 5 for 48 hour,and decreased from 34.37 % to 16.43% for 72 hour(P<0.05).RT-PCR showed that the expression of Ect2 mRNA was(0.77±0.06) and(0.85±0.04)in the group of 0.1 nmol/L and 1 nmol/L of annexin 5 respectively.Compared with control group(0.67±0.06),the expression of Ect2 mRNA increased by 14.9%(P<0.05) and 26.9%(P<0.01) respectively.However,no significantly enhanced expression of Ect2 mRNA in the group of 10 nmol/L of annexin 5 was shown.The results of western bolt showed 20.9% increase of Ect2 protein in group of 1 nmol/L of annexin 5 compared with control group [(1.50±0.15) vs(1.24±0.07),P<0.05],but no significantly increase of Ect2 protein was shown in the groups of 0.1 and 10 nmol/L of annexin 5.Conclusion Annexin 5 promoted rat Leydig cells proliferation by increasing the transition of the cells from G2 to M phase.The expressions of both gene and protein of Ect2 in rat Leydig cells were regulated by annexin 5.The regulation of annexin 5 for proliferation of rat Leydig cells may realize through promoting the expression of Ect2.