小鼠睾丸支持细胞的分离培养与鉴定

Isolation and identification of Sertoli cells from mouse testis

目的：分离培养小鼠睾丸支持细胞并进行鉴定。方法：取18～20d雄性小鼠的睾丸，酶消化法原代培养。用RT—PCR的方法克隆带有锌指结构域的支持细胞基因SERZ，与pcDNA3．0连接后构建重组质粒pcDNA3．0-SERZ。用KpnI和Xba1分别酶切质粒pcDNA3．0-SERZ，获得线性化模板进而合成探针。原位杂交检测SERZ mRNA在培养细胞中的表达；用RT—PCR的方法检测雄激素结合蛋白（ABP）在支持细胞中的表达。结果：支持细胞多为多边形，核呈三角形或不规则形，染色浅，核仁明显，细胞完全铺开，成膜状，相邻细胞之间交织连接，细胞纯度为（85．1±2．5）％。SERZ mRNA在培养细胞的胞质中高水平表达。RT—PCR结果表明我们分离的支持细胞能够表达ABP mRNA。结论：我们成功分离和鉴定了小鼠睾丸支持细胞，纯度为（85．1±2．5）％。

Objective： To isolate and identify Sertoli cells from mouse testis. Methods： Testis were isolated from male mouse aged 18-20 days old and were cultured by enzymatic digestion. The Sertoli cell gene with a zinc finger domain （SERZ） was obtained by RT-PCR and was inserted into pcDNA3.0 to construct recombinant plasimid pcDNA3.0-SERZ, pcDNA3.0- SERZ was then cleaved by restriction endonuclease Kpn I and Xba I to obtain the linear templets for preparation of the probes. The expression of SERZ mRNA in the cultured cells was analyzed by in situ hybridization with the prepared probes. The expression of androgen binding protein （ABP） mRNA in the cultured cells was detected by RT-PCR. Results： Under light microscope, most Sertoli cells were polygonal and were completely extended,mimicking a membrane. The nuclei were triangular or irregular, weakly stained and with obvious nucleoli. The neighbouring cells were interlaced with one another and the cell purity was （85.1± 2.5） %. SERZ mRNA was highly expressed in the cytoplasm of the cultured cells. RT-PCR showed that ABP mRNA was expressed in the cultured cells. Conclusion： We have successfully isolated and identified Sertoli cells from mouse testis, with the cell purity being （85.1±2. 5）%.