大鼠睾丸间质细胞体外培养不同时间3β-羟基类固醇脱氢酶的表达

Expression of 3β-Hydroxysteroid Dehydrogenase in Rat Leydig Cells Cultured by Differential Adhesion In Vitro

目的探讨通过差速贴壁法获得的7天龄、3周龄大鼠睾丸间质内细胞的细胞群体构成、体外培养不同时间3β-羟基类固醇脱氢酶(3β-HSD)的表达水平变化,以及细胞睾酮分泌功能变化。方法联合应用胶原酶消化、不锈钢滤网过滤及差速贴壁法获得7天龄、3周龄雄性Wistar大鼠睾丸间质组织内细胞,贴壁细胞以DMEM/F12培养液培养,分别对原代培养2h、4d细胞进行3β-HSD免疫化学染色及流式细胞分析,原代培养细胞同时给予人绒毛膜促性腺激素(HCG)刺激,测定HCG刺激组和非刺激组各代细胞培养液上清中睾酮水平及其对HCG刺激的反应。结果7天龄、3周龄鼠差速贴壁法获得的睾丸间质内细胞群经流式细胞鉴定,3β-HSD阳性细胞所占比例分别为(5.4±1.2)%、(59.2±3.2)%;培养4d后,两组3β-HSD阳性细胞比例分别为(93.6±1.2)%、(95.4±3.2)%。两组原代培养细胞均有睾酮生成功能,在HCG刺激下睾酮分泌均明显上升,7天龄组培养细胞睾酮分泌高峰迟于3周龄组。结论差速贴壁法获得的7天龄、3周龄大鼠睾丸间质细胞群中均含有部分Leydig干细胞(Stem Leydig cells,SLCs),SLCs在体外培养过程中逐渐分化,表达3β-羟基类固醇脱氢酶,并产生睾酮。

Objective To investigate the constitution of cells population, changes in 3β-hydroxysteroid dehydrogenase （3β-HSD） expression and androgen productivity in rat Leydig cells cultured by differential adhesion during 4 days primary culture. Methods Testes from 7 days and 3 weeks old rats were harvested, then Leydig cells were isolated by using collagenase dispersion, stainless steel mesh infiltration and differential adhesion. 3β-HSD expression in cultured cells was observed by immunohistochemistry and flow Cytometry analysis immediately after cell isolation and 4 days later. Testosterone level in isolated Leydig cells with or without HCG stimulation was also tested. Results In 7 days group and 3 weeks old group 3β-HSD positive rates in cultured cells were 5.4%±1.2% and 59.2%±3.2% respectively after isolation immediately. 4 days after culture, most of the cultured cells （93.6%±1.2% in 7 days old group, 95.4%±3.2% in 3 weeks old group） became 3β-HSD positive. Testosterone level, increased by HCG stimulation, in cultured cells from both groups. Conclusion Leydig cells obtained by differential adhesion from 7 days and 3 weeks old rats consist Stem Leydig cells （SLCs）. These SLCs were differentiated and gained 3β-HSD activity during cell culture.