Abstract: The study aimed to study the characterization of spermatogonial stem cells（SSCs）from Guangdong Small⁃spotted Pig testis in vitro after separation，purification and in vitro culture. The investigation employed enzyme digestion， density gradient centrifugation， and differential adhesion methods for cell enrichment， and cell immunofluorescence staining was used to assess separation efficiency，cell proliferation and differentiation. The results indicated that the combination of three separation methods effectively improved the purity of SSCs，which were successfully proliferated in vitro，forming distinct cell colonies by day 14，then cell colonies proliferation was significantly increased by day 21. Immunostaining showed that the SSCs maker，UCHL⁃1 was expressed highly within 28 days，while the expression of C⁃kit on day 56 indicated cell differentiation.

Key words: Guangdong Small?spotted Pig, Testis, Spermatogonial stem cells, Immunofluorescence staining, UCHL?1

摘要： 该研究采用酶消化法、密度梯度离心法和差速贴壁方法对广东小耳花猪的精原干细 胞进行富集纯化，并通过细胞免疫荧光染色检测分离效果、细胞增殖和细胞分化程度。结果表 明，三种分离方法结合使用可以有效提高精原干细胞的纯度，在饲养层细胞的支持下，精原干 细胞成功在体外增殖，在14 d形成明显的细胞集落，21 d时集落大量形成，细胞增殖加快；免 疫染色结果显示，细胞在培养28天内精原干细胞标记蛋白UCHL?1高表达，但在培养56 d后， 部分细胞已表达酪氨酸激酶受体（KIT proto?oncogene receptor tyrosine kinase，c?KIT），表明精原 干细胞已出现分化。该研究将为广东地方猪保种和基于精原干细胞遗传修饰的种质创新提供研究基础。

关键词: 小耳花猪, 睾丸, 精原干细胞, 细胞免疫染色, UCHL?1