猪雄性生殖干细胞的分离培养及鉴定

本试验旨在探索猪雄性生殖干细胞(mGSCs)体外分离、培养的适宜条件,建立猪雄性生殖干细胞体外培养体系。采用两步酶消化法对新生小猪睾丸生殖干细胞进行了体外分离和初步的培养鉴定,并利用层黏连蛋白和明胶的不同贴壁特性,比较2种差易贴壁分选方法的富集效果,并对传代后的干细胞培养1周后进行碱性磷酸酶染色鉴定,通过免疫荧光技术检测培养细胞是否表达干细胞标志蛋白OCT-4。试验结果表明,层黏连蛋白更适用于猪生殖干细胞的富集、培养,细胞分选效率及增殖生长明显优于采用明胶分选的方法。培养的mGSCs拥有与小鼠mGSCs相同的形态、增殖及表达特征。鉴定结果显示,生长细胞克隆碱性磷酸酶染色呈阳性,支持细胞碱性磷酸酶染色呈阴性;培养的生殖干细胞克隆表达转录蛋白OCT-4,而饲养层支持细胞OCT-4抗体染色则呈阴性。结果表明培养的干细胞克隆仍保持较好的干细胞活性,保持正常的自我复制和分化潜能,初步建立了生殖干细胞培养体系。

Isolation,Culture and Identification of Porcine Male Germline Stem Cells

This study was aimed to explore the suitable conditions for the isolation,culture of pig male germline stem cells, and establish the in vitro culture system. The cells were isolated from the neonatal pig test by the two step enzyme digestion method, and cultured for further identification, compared the enrichment efficiency of cells on laminin and gelatin due to the different adherence velocity. And then the cells were identified for the alkaline phosphatase activity and the expression of the stem cell marker protein OCT-4. The results showed that laminin was more suitable for the enrichment and growth of porcine germline stem cells. The enrichment efficiency and proliferation rate of cells using laminin method was obviously superior to which using gelatin sorting. The cultured mGSCs had the similar morphology and proliferation characteristics to mGSCs of mouse. The expression of OCT-4 was positive in target cells, while it was negative in feeder cells-sertoli cells. The target cells showed strong expression of alkaline phosphatase, while nothing was detected in feeder cells-sertoli cells. The results showed that, we have established the preliminary culture system of porcine germline stem cells, with well maintained stem cell activity, normal replication function and differentiation potentials.