【摘要】 目的运用蛋白质组学方法,寻找大鼠精子发生相关蛋白质,找到目标蛋白质后,进一步用免疫组织化学方法做定位研究。方法用重力沉降法(STAPUT法)从9 d龄雄性大鼠睾丸中分离出A型精原细胞,从成年的雄性大鼠睾丸中分离出粗线期精母细胞、圆形精子细胞。分别提取这3种细胞的总蛋白质,进行双向电泳。对所得双向电泳凝胶扫描,分析并找出差异蛋白质。对挑选出的差异蛋白质做质谱分析,发现在这3种不同细胞的表达上有明显差异的蛋白。进一步做睾丸免疫组织化学组织定位、定量研究。结果双向电泳结果显示,HSP70-2在粗线期精母细胞、圆形精子细胞中表达量较高, 在A型精原细胞则表达量极少。HSP70—2在粗线期精母细胞中表达量最大。用HSP70—2抗体对大鼠睾丸组织切片行免疫组织化学研究,发现粗线期精母细胞、Sertoli细胞、Leydig细胞有阳性颗粒反应。HSP70—2主要表达于细胞质。结论 HSP70—2在精子发生过程中有明显的差异表达,推测其对精子发生起重要调节作用。更多还原

【Abstract】 Objective: The two-dimensional electrophoresis (2-DE) method was used for screening the stage-specific expression proteins during rat spermatogenesis. Then the immunohistochemical study was used for the specific protein localization. Methods: The highly enriched spermatogonia, pachytene spermatcoytes and round spermatids were isolated by STAPUT method (sedimentation velocity at unit gravity, with 2%-4% BSA gradient in DMEM/F12 medium) to get the total proteins from the three kinds of cells. The protein expression profiles in the three kinds of cells were respectively analyzed by two-dimensional electrophoresis. By means of 2-DE, HSP70-2 protein was exclusively detected. Then the distribution of HSP70-2 immunoreactivity in rat testicular tissues was investigated with a specific anti-HSP70-2 anti body by immunochemistry method. Results: The results of 2-DE showed that HSP70-2 expression was stronger in pachytene spermatcoytes than in round spermatids. The subcellular localization of HSP70-2 was mainly in cytoplasm. The positive immunoreactivity was mainly located in pachytene spermatcoytes, Sertoli cell and Leydig cell. Conclusion: These results suggest that HSP70-2 is a stage-specific protein and plays an important role in spermatogenesis.更多还原