金毛犬睾丸成纤维细胞的分离、培养及鉴定

Isolation, culture and identification of testicular fibroblasts in golden retriever

为获得金毛犬睾丸组织成纤维细胞,试验采用胰蛋白酶消化睾丸组织,对成纤维细胞进行体外分离、培养及冻存,用RT-PCR法鉴定成纤维细胞特异基因波形蛋白(vimentin),观察比较第2代(P2)及第5代(P5)细胞生长曲线、群体倍增时间、克隆形成率。结果表明:用胰蛋白酶法消化睾丸组织获得的成纤维细胞呈典型的纺锤状,其可以表达特异基因vimentin,P2及P5细胞生长曲线均呈"S"型, P2及P5成纤维细胞群体倍增时间差异不显著(P>0.05);P2成纤维细胞的克隆形成率显著高于P5(P<0.05)。说明通过胰蛋白酶法消化睾丸组织可高效分离和获得犬睾丸成纤维细胞。

The aim of the present study was to obtain fibroblasts from the testicular tissues of golden retriever.The testicular tissues were digested by trypsin method and the fibroblasts were isolated,cultured and frozen in vitro.Fibroblast specific gene vimentin was identified by RT-RCR method,the cell growth curve,population doubling time and clone formation rate of generation 2(P2)and generation 5(P5)were observed and compared.The results showed that the testicular fibroblasts had a typical spindle shape and expressed vimentin.The growth curves of the second and fifth generation cells were S-shaped,and there was no significant difference in population doubling time of the second and fifth generation fibroblasts(P>0.05).The clone formation rate of fibroblasts in the second generation was significantly higher than that in the fifth generation(P<0.05).The results indicated that canine testicular fibroblasts could be isolated and obtained by digesting testicular tissue with trypsin efficiently.