小鼠睾丸引带细胞的培养及己烯雌酚对培养睾丸引带细胞的影响

Effects of Diethylstilbestrol on Cultured Testis Gubernacular Cells in Mice

目的：探讨小鼠睾丸引带细胞体外培养的有效方法,并进行形态学观察。研究经典的外源性雌激素己烯雌酚（DES）体外对小鼠睾丸引带发育的影响。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并进行细胞培养。台盼蓝染色检测细胞存活率,HE染色观察细胞形态。传代培养并随机分为空白对照组、溶剂对照组（DMSO组）和实验组（DES 1~4组）共6组。溶剂对照组加入DMSO,实验组分别加入0.01、0.10、1.00以及10.00μg/ml DES。分别于培养12、24、48 h后进行细胞形态学观察,CCK-8法检测睾丸引带细胞的生长情况。结果：培养睾丸引带细胞大部分为成纤维细胞型,有少数的上皮样细胞。原代细胞的存活率为85%~90%。在不同剂量DES作用后的3个不同时间段里,细胞增殖性的检测结果存在时间-剂量效应的显著差异（P〈0.01）。结论：睾丸引带细胞可经一定的方法进行体外培养,外源性雌激素对睾丸引带细胞的生长有抑制作用,且呈现一定的时间-剂量效应。对培养睾丸引带细胞影响的研究是外源性雌激素影响生殖系统发育研究的一条有效的睾丸外途径。

Objective： To establish a primary culture of the testis gubernacular cells of Kunming mice,observe the morphological characteristics of the cells,and explore the effects of exogenous estrogens（EEs） on the development of the testis gubernacula in vitro.Methods： We removed the gubernacula from 3-day-old mice with the surgical magnifier and cultured the gubernacular cells.Then we detected the cell viability by trypan blue and cell morphology by HE staining.The subcultured cells were randomly divided into a blank control,a DMSO（0.1%,v/v） control,and 4 experimental groups（given 0.01,0.10,1.00 and 10.00 μg/ml of diethylstilbestrol dissolved in DMSO,respectively）.After treated for 12,24 and 48 hours,the gubernacular cells were observed for morphological changes and proliferation inhibition by CCK-8.Results： Most of the cultured gubernacular cells were fibroblasts,and a few were epithelioids.The primary cells showed a viability of 85%-90%.Dose-and time-dependent inhibition of cell proliferation was found in the four experimental groups at three different times,with statistically significant differences（P 0.01）.Conclusion： Gubernacular cells can be cultured in vitro.EEs inhibit the proliferation of gubernacular cells in a dose-and time-dependent manner.An insight into the effects EES on cultured gubernacular cells is an effective approach to the study of their influence on the development of the reproductive system.