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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。
OBJECTIVE: To screen and identify the self-proliferation related protein factors of spermatogonial stem cells (SSCs) in mice and to explore their effects on the proliferation and differentiation of SSCs. Methods: By establishing an effective animal model and using proteomics methods such as two-dimensional electrophoresis and mass spectrometry, we screened and identified the protein factors that may be related to the self-proliferation of mouse SSCs. Cloned the genes of the selected proteins and compared them with that of green fluorescent protein Of the gene fusion to construct vector pEGFP-stathmin. Transfect mouse spermatogonia and testicular support cells cultured in vitro to make them overexpressed in the cells. The cells were counted by MTT method after 1, 3 and 5 days respectively. The expression of the selected proteins in spermatogonia was analyzed by in situ hybridization. RESULTS: Four proteins that might be related to the self-proliferation of SSCs were screened successfully, and stathmin was one of them. The number of spermatogonia in stathmin transfection group was significantly higher than that in empty vector transfection group and normal control group (P <0.05) on the 2nd, 4th, and 6th day after transfection of fusion gene. In situ hybridization results showed that stathmin mainly Expressed in spermatogonia, but weakly expressed in spermatocytes. Conclusion: stathmin can promote the proliferation of spermatogonia in mice, and this effect is very likely to affect the differentiation of spermatogonia to spermatocytes.