小鼠胚胎睾丸Leydig细胞培养、纯化及其功能研究

Isolation,Cultivation,Identification and Functional Study of Fetal Mice Leydig Cells in vitro

目的:探讨小鼠胚胎睾丸Leyd ig细胞体外培养、鉴定、纯化的方法,并进行形态学观察、分泌睾酮能力等生物学特性检测。方法:选择胎龄16 d的胎鼠睾丸,0.03%胶原酶Ⅰ消化,3β-羟基类固醇脱氢酶(HSD)染色鉴定及纯度测定,锥虫蓝染色检测细胞活率。放免法测定Leyd ig细胞不同培养时间及密度下分泌睾酮的水平。结果:Leyd ig细胞培养前和培养72 h后纯度分别为(45.10±1.66)%和(81.17±2.32)%;培养液中可检测到睾酮,睾酮水平与Leyd ig细胞数、培养时间相关,单个Leyd ig细胞睾酮分泌能力逐日下降。结论:该法分离的胚胎Leyd ig细胞纯度较高,生长性好,保持增殖和分泌睾酮的生物学特性,可应用于相关的研究。

Objective： To explore the methods of isolation, cultivation, purification, identification of the fetal mice testis Leydig cell and to observe its biological characteristics in vitro. Methods ： Leydig cells were isolated by 0.03 % collagenase （ type Ⅰ） from fetal mice testis and cultured in 1）MEM/F12 medium. The identity and purity of Leydig cell were assessed by 3β-hydroxysteroid dehydrogenase △4-△5 isomerase （313-HSD）. Cell viability was measured by trypan blue. Testosterone level in the medium of cultured Leydig cells was measured in various culture phases and cell density by radioimmunoassay. Results： The purity of Leydig cell was （45.10± 1.66 ）% before culture, and （81.17 ±2.32）% 72 h after culture. The level of testosterone secreted by Leydig cells could be detected in the medium and its level was associated with the density and time of cultured Leydig cells. The secretion capacity of testosterone by single Leydig cell decreased gradually during the culturing period. Conclusion ： The fetal Leydig cells isolated from fetal mice testis have high purity. It can be cultured and kept the secretion ability of testosterone for a few days in vitro. This system can provide a valuable model for further study on the cellular function of the Leydig cells of fetal mice.