胰岛素样因子3对体外培养的小鼠睾丸引带细胞增殖和收缩活性的影响

Effect of Insulin-Like Factor 3 on Proliferation and Contraction of Mouse Gubernacular Cells in vitro

目的探讨胰岛素样因子3(INSL3)对体外培养的小鼠睾丸引带细胞增殖和收缩活性的影响。方法手术放大镜解剖出3日龄雄性昆明小鼠的睾丸引带组织,进行原代细胞培养后传代。将传代细胞随机分为正常对照组和实验组,实验组加入INSL3,浓度分别为3.3×10-3μmol.L-1、3.3×10-4μmol.L-1、3.3×10-5μmol.L-1及3.3×10-6μmol.L-1,分别持续作用12 h、24 h、48 h,利用细胞计数试剂盒(CCK-8)检测其细胞增殖情况;细胞免疫荧光和流式细胞术检测其细胞纤维状肌动蛋白(F-actin)的结构变化及表达情况。结果不同浓度INSL3作用后的不同时间点,检测细胞增殖和收缩活性的相关指标,结果均存在时间-剂量效应,且差异有统计学意义(P<0.05)。正常对照组及各实验组细胞均呈持续性增殖,24 h后增殖更明显。与正常对照组比较,INSL3可刺激睾丸引带细胞骨架重塑,细胞周边肌动蛋白丝带增多,胞质中微丝F-actin明显粗大、变长,细胞中F-actin表达量增加。各实验组中以3.3×10-3μmol.L-1组变化尤其明显。结论 INSL3对睾丸引带细胞增殖和收缩活性有直接促进作用,可能参与睾丸引带发育甚或睾丸下降过程的调节。

Objective To investigate the effect of different doses of insulin-like factor 3（INSL3） on the proliferation and contraction of mouse gubernacular cells in vitro. Methods The gubernaculum from 3-day-old mice with the surgical magnifier were dissected and the gubernacular cells were cultured.The subcultured cells were randomly divided into normal control group and 4 experimental groups（3.3×10^-3 μmol·L^-1,3.3×10^-4 μmol·L^-1,3.3×10^-5 μmol·L^-1 and 3.3×10^-6 μmol·L^-1）.After treated for 12 hours,24 hours and 48 hours,the proliferation of gubernacular cells were quantitated by cholecystokinin octapeptide（CCK-8）,and the structural changes and the expressions of F-actin in gubernacular cells were detected by immunofluorescence and flow cytometry methods. Results Dose-and time-depen-dent inhibition of cell proliferation and contraction were found in the 4 experimental groups at different time points,with statistically significant differences（P0.05）.Gubernaculum cells showed persistent proliferation in the normal control group and experiment groups,especially after 24 h.Compared with the normal control group,INSL3 could stimulate the increase of the actin filaments and up-regulate the expression of F-actin in gubernaculum cells,and the filaments of F-actin in the cytoplasm elongated became more stout,whose changes were most evident for the 3.3×10^-3 μmol·L^-1 group. Conclusions INSL3 can directly improve the proliferation and contraction of mouse gubernacular cells,which may be a possible mechanism of gubernacular development and descent.