摘要
目的:筛选并鉴定小鼠精原干细胞(spermatogonial stem cells,SSCs)自增殖相关的蛋白因子,并深入探讨其对SSCs自增殖及分化的影响。方法:通过建立有效的动物模型,运用双向电泳、质谱分析等蛋白组学的方法,筛选并鉴定与小鼠SSCs自增殖可能相关的蛋白因子;克隆所筛选蛋白的基因,使其与绿色荧光蛋白的基因融合,构建载体pEGFP-stathmin。转染体外培养的小鼠精原细胞及睾丸支持细胞,使其在该细胞中过表达。分别培养1、3和5d后,以MTT法对细胞进行计数;通过原位杂交,对所筛选蛋白的基因在精原细胞中的表达进行分析。结果:成功筛选了4种与SSCs自增殖可能相关的蛋白,stathmin即是其中一种。在转染融合基因后培养的第2、4、6天时,stathmin转染组精原细胞数显著高于空载体转染组以及正常对照组(P<0.05);原位杂交结果显示,stathmin主要表达在精原细胞中,而在精母细胞中有弱表达。结论:stathmin对小鼠精原细胞增殖具有促进作用,该影响极有可能作用于精原细胞向精母细胞的分化阶段。
[Abstract] Objective：To screen proteins related to spermatogonial self-renewal and to further explore their effect on spermatogonia. Methods：Ten-day-old male Kunming mice were surgically rendered cryptorchid. The differently expressed proteins between experimental cryptorchid mice and sexually mature mice were screened and compared by proteomic tech-niques. As one of these proteins, stathmin were screened and identified, stathmin gene was cloned, and fused with pEGFP. And the fused gene was transfected into spermatogonia and sertoli cells. The number of germ cells in the plate was counted by MTT. In situ hybridization histochemistry for stathmin was performed. Results ： Four proteins were successfully screened and compared by proteomic techniques, one of which was stathmin. After transfection green fluorescence appeared in the nucleus of spermatogonia rather than in sertoli nucleus. On the 2nd, 4th and 6th day after transfectian, the number of cells in the plate transfected with pEGFP-stathmin was langer than in the non-transfed plate or that of the group transfected with pEGFP vector only. In situ hybridization histochemistry showed that stathmin was expressed in spermatogonia and primary spermatocyte. Conclusion：Stathmin stimulates germ cell proliferation in mice testis and mainly relates to the stage from spermatogonia to primary spermatocyte.
出处
《军事医学科学院院刊》
CSCD
北大核心
2009年第5期412-415,共4页
Bulletin of the Academy of Military Medical Sciences
基金
国家自然科学基金资助项目(No.30972090)
河南省自然科学基金资助项目(No.092300410200)
黄淮学院青年骨干教师资助计划
后勤科研项目(08ZL030)
