表皮生长因子对大鼠睾丸间质干细胞增殖和分化的影响研究

Effect of epidermal growth factor on proliferation and differentiation of rat Leydig cells

目的研究表皮生长因子对大鼠睾丸间质干细胞增殖和分化的影响。方法二甲磺基乙烷单次腹腔注射成年雄性大鼠(75 mg/kg),7 d后,分离曲细精管,构建曲细精管体外培养模型,进行处理:①表皮生长因子处理24 h,试剂盒进行5-乙炔基-2′脱氧尿嘧啶核苷(EdU)标记,Image-Pro Plus软件统计分析睾丸间质干细胞增殖情况;②表皮生长因子处理3 d后,换为含LH培养基继续培养至21 d,收集培养基上清液,放射免疫法检测上清液睾酮的分泌情况,同时收集曲细精管提取RNA,荧光定量PCR检测睾酮合成通路相关基因的表达情况,综合分析睾丸间质干细胞在表皮生长因子作用下的分化情况。结果表皮生长因子能够显著地促进睾丸间质干细胞的增殖;曲细精管培养基上清液的睾酮水平明显上升,并且在睾酮合成通路上Star、Cyp11a1和Hsd3b1经过表皮生长因子诱导后,在mRNA水平上均出现了明显的上调,表明睾丸间质干细胞出现了分化。结论表皮生长因子能够诱导睾丸间质干细胞的大量增殖和向睾丸间质细胞系的分化。

Objective To study the effect of epidermal growth factor(EGF)on the proliferation and differentiation of rat Leydig cells.Methods The male rats were treated 1 week prior to the beginning of experimentation with ethane dimethanesulfonate(EDS,75 mg/kg body weight,by i.p.injection).We used the established organ culture model of rat seminiferous tubules to examine the regulation of EGF during SLC proliferation using EdU staining after incubated for 24 h;We examined the regulation of EGF during SLC differentiation in organ culture model of rat seminiferous tubules,the EGF group was incubated for 3 days and then treated with DMEM/F12-ITS media containing LH for up to 21 days and the culture media from the seminiferous tubules were collected and testosterone concentrations in the media were measured using I 125-based RIA;Real-time PCR was performed to examine mRNA levels of steroidogenic genes and the transcription factor Star in seminiferous tubules after treatment with EGF and LH.Results EGF significantly promoted the proliferation of stem Leydig cells and also induced steroidogenic enzyme gene expression and EGF promoted testosterone production in seminiferous tubules in vitro.Conclusion EGF is capable of inducing massive proliferation of testicular mesenchymal stem cells and differentiation into Leydig cells.