RXFP2在己烯雌酚染毒小鼠睾丸引带细胞中的作用机制研究

RXFP2 participates in diethylstilbestrol-mediated effects on morphology and proliferation of mouse gubernaculum cells

目的通过检测己烯雌酚(DES)对小鼠睾丸引带细胞形态与增殖性影响过程中松弛素家族肽受体2(RXFP2)的表达水平,探讨RXFP2在外源性雌激素影响小鼠睾丸引带细胞中的可能作用机制。方法小鼠睾丸引带组织进行细胞培养,传代后随机分为正常组、溶剂对照组及实验组,实验组分别加入DES终浓度为0.01、0.10、1.00、10.00μg/m L作用48 h。观察细胞形态变化,CCK-8检测其增殖情况,应用免疫荧光和Western Blot对小鼠睾丸引带细胞中RXFP2进行定位和蛋白定量分析。结果正常组细胞呈成纤维细胞型生长,同源性好。各实验组随DES剂量增加细胞数及细胞生存率均下降,细胞增殖明显受抑制,失去正常形态,胞浆收缩,胞体形态不规则。免疫荧光显示RXFP2高表达于细胞膜上。蛋白定量分析发现各实验组RXFP2水平与正常组相比表达降低,且差异有统计学意义。结论DES可影响小鼠睾丸引带细胞形态与增殖性以及RXFP2蛋白的表达,推测DES可能通过影响RXFP2信号系统介导睾丸引带的发育,进而影响睾丸的下降。

Objective To investigate the effects of diethylstilbene(DES)on the morphology and proliferation of mouse gubernaculum cells and explore the role of RXFP2 in these pathological process.Methods Mouse gubernaculum primary cells were isolated and cultured,then they were randomly divided into three groups including the normal group,solvent control group and the experimental group.In the experimental group,the cells were treated with DES with final concentration of 0.01,0.10,1.00,10.00μg/ml.The morphological changes of the cells were observed,and the proliferation of the cells was detected by cck-8.The localization and expression analysis of RXFP2 in gubernaculum cells were measured by immunofluorescence and Western Blot.Results The cells in the normal group exhibited relatively uniform spindle-shaped in appearance.With the increase of DES dose,cell number and cell survival rate in all experimental groups were decreased.Simultaneously,cell proliferation was significantly inhibited,and cell morphology presented normal morphology decrease,cytoplasmic contraction,and irregular cell morphology.Immunofluorescence showed that RXFP2 was highly expressed in the membrane of cells.Protein quantitative analysis showed that RXFP2 expression was remarkably decreased in each experimental group compared with that in the normal group,which was statistically significant.Conclusion DES can affect the morphology,proliferation and RXFP2 expression of mouse gubernaculum cells.These findings implied that DES might affect on the development of gubernaculum by regulating the RXFP2 signaling pathway.