锌指蛋白185在小鼠睾丸间质细胞的表达研究

Expression of ZNF185 in Leydig cells of mouse testis

目的优化睾丸间质细胞分离纯化方法,研究锌指蛋白(zinc finger protein,ZNF)185在间质细胞中的表达。方法选用4-5周雄性小鼠,取睾丸,Ⅳ型胶原酶消化分离间质细胞,差速贴壁法纯化后,3β-羟基类固醇脱氢酶染色法鉴定细胞纯度,PCR、Western blot和免疫荧光法研究ZNF185在间质细胞中的表达。结果成功分离纯化小鼠睾丸间质细胞,有纯化组细胞的纯度(x珋=98%)显著高于无纯化组(x珋=22%)(P<0.001)。PCR和Western blot显示,ZNF185在间质细胞中高表达。免疫荧光显示,ZNF185定位于间质细胞的细胞质。结论通过该方法可获得高纯度间质细胞;ZNF185主要表达于间质细胞的细胞质,为进一步研究该蛋白的功能提供了实验基础。

Objective To optimize the method of isolating and purifying Leydig cells of male mouse,and investigate the expression of ZNF185 in Leydig cells. Methods The 4-5-week-old male mice were used,and testes were collected and digested with type Ⅳ collagenase,purified by differential attachment of cells,and identified by 3β-hydroxy-steroid-dehydrogenase staining method. The expression of ZNF185 was detected by PCR,Western blot and immunocytochemical analysis. Results The Leydig cells of mouse testis were successfully isolated and purified,and the purity in purification group（ χ^2= 98%） was significently higher than that in non-purification group（ χ^2= 22%）（ P〈0. 001）. PCR and Western blot results showed that ZNF185 was highly expressed in Leydig cells. ZNF185 was localized in the cytoplasm of Leydig cells by immunocytochemical analysis. Conclusion This method can successfully produce highly purified Leydig cells. ZNF185 is mainly localized in cytoplasm of Leydig cells,which provides evidences for the further investigation of its function.