家兔精原干细胞的分离培养与鉴定

Isolation,culture,and identification of rabbit spermatogonial stem cells

为了探索家兔精原干细胞(SSCs)体外分离、培养的适宜条件,建立家兔SSCs体外培养体系,试验采用两步酶消化法从家兔睾丸中分离SSCs,并采用多次差异贴壁法对所培养的SSCs进行纯化,再通过碱性磷酸酶(AKP)染色、RT-PCR及免疫细胞化学染色进行检测。结果表明:所培养的细胞形态为圆形;AKP染色呈阳性,支持细胞呈阴性;培养细胞中有SSCs特异性基因Ngn3及Oct4的表达;免疫细胞化学染色检测到干细胞表面标记CD9的存在。说明试验所培养细胞克隆为家兔SSCs且具有良好的干细胞活性,以及正常的自我更新和分化潜能。

To explore the suitable conditions for the in vitro isolation and culture of rabbit spermatogonial stem ceils （ SSCs）, and to establish the in vitro culture system. The two step - enzyme digestion method was used to isolate the SSCs from rabbit testis, and then the multiple differ- ential adhesion method was used to purify the cultured SSCs, which were tested using alkaline phosphatase （AKP） staining, RT - PCR and im- munocytochemistry techniques. The results showed that the cultured cells were round in morphology. Positive AKP staining was detected, and the sertoli cells were negative. The expressions of specific gene Ngn3 and Oct4 from SSCs were involved in these cultured ceils, and the exist- ence of stem cell surface marker CD9 was tested by using immunoeytochemistry techniques. The results indicate that the cultured cells are cloned as the rabbit SSCs with good stem cell activity, normal self - renewal and differentiation potentials.