大鼠精原细胞的分离纯化

Isolation and purification of spermatogonium in rat

目的:探讨大鼠精原细胞的分离纯化. 方法:采用酶消化法制备10 d Wister大鼠睾丸单细胞悬液;Percoll液不连续密度梯度分离精原细胞;用贴壁速度的差异纯化精原细胞.光电镜观察精原细胞的形态结构特征;应用酪氨酸蛋白激酶(c-kit)免疫化学鉴定精原细胞. 结果:精原细胞主要分布在28%～36%(Ⅱ带)间的Percoll梯度;分离纯化的精原细胞形态结构与组织切片上精原细胞一致,其纯度达55.68%;c-kit在精原细胞呈阳性表达,在睾丸体细胞呈弱阳性或阴性表达.结论:利用酶消化法、Percoll液不连续密度梯度及细胞贴壁速度的差异,分离纯化的精原细胞满足体外实验要求;精原细胞表达特异的c-kit受体.

Objective: To study isolation and purification of rat spermatogonium. Methods: Digestions by enzymes were used to prepare germ cell suspension of Wister rat at 10 days;Percoll discontinue density gradient centrifugation and differentiation of velocity of cells sticking to the wall in vitro were used to isolate and purify spermatogonia respectively; Morphological and structural features of spermatogonia in rat at 10 d of age were observed by light and transmission electron microscope; Spermatogonium was identified by c-kit (tyrosine protein kinase) immunochemistry. Results: Spermatogonia were mainly distributed in percoll gradient between 28%~36%, Their morphological and structural features were consistent with that in testis sections of 10 days old rat. Purify of spermatogonia was 55.68% after purified. Spermatogonia were c-kit-positive, but c-kit expression of testic body cells was weak positive or negative. Conclution: Spermatogonia isolated and purified from rat at 10 days by digestions with enzymes, Percoll discontinue density gradient centrifugation and differentiation of velocity of cells sticking to the wall in vitro could satisfy the needs of test in vitro. Spermatogonia expressed specific c-kit receptor.