精原干细胞动态标志物的筛选与确定

While significant progress has been made in the research on markers of spermatogonial stem cells (SSCs), the ideal specific markers for SSCs have not been identified yet. It remains to be clarified whether the expression of SSC markers is time-dependent, or specific markers express in different stages. As a technique for proteomic analysis, the application of two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has not been reported in the dynamic analysis of SSC markers. Based on our previous research on the surface markers, separation and purification, culture in vitro and transplantation of SSCs, we will separate and purify SSCs of mouse at various ages, observe the comprehensive proteome profile of SSCs dynamically by two-dimensional electrophoresis and MALDI-TOF-MS, then screen differential protein expression or discover new proteins in mouse SSCs through the bioinformatics proteomics database. We will then produce recombinant protein through molecular cloning methods and prepare the corresponding monoclonal antibody, employ the relevant antibody to perform immunohistochemistrical detection on mouse testicular tissue slices in succession. In order to observe the proliferation and differentiation of SSCs in vivo,which have been sorted by immunomagnetic beads and the relevant antibody, transplant the purified SSCs into the donor seminiferous tubuli by microinjection technique. We will verify specific markers of mouse SSCs at different ages by those morphological means and observe whether their expression is time-dependent. In order to identify specific markers of spermatogonial stem cells, we will apply the candidate markers which have been screened in mouse SSCs to human normal testicular tissue samples of different ages, and observe the protein expression, as well as phasic change in human SSCs.

尽管对精原干细胞（SSCs）标志物研究已取得一些进展，迄今尚未找到理想的SSCs标志物，是否SSCs标志物的表达具有时相性，在特定阶段表达特定标志物，需要明确。双向电泳及基质辅助激光解析飞行时间质谱系统作为蛋白质组学分析技术，目前尚没有采用该方法对SSCs标志物进行动态分析研究的报道。我们在前期对SSCs表面标志物、分离纯化、体外培养及移植研究的基础上，拟将不同年龄段小鼠SSCs分离纯化后，通过此分析系统动态观测各年龄段SSCs蛋白表达谱，运用生物信息学蛋白质组数据库筛选差异性蛋白或发现新的蛋白。用分子克隆的方法生产重组蛋白，制备相应单克隆抗体，通过免疫组织化学结合曲细精管微注射移植技术，通过形态学方法映证各年龄段小鼠SSCs特异性标志物，并观测其时相性。在不同年龄段人体正常睾丸组织标本中，观测在小鼠SSCs中所候选的标志物在人体睾丸组织中的表达情况，期望能筛选到SSCs新的特异性标志物。

精原干细胞移植对男性不育的治疗具有潜在的临床应用价值，尤其对青春期前接受大剂量化疗致生精功能严重受损的非梗阻性无精子症患者，具有独特的临床应用价值。只要在非梗阻性无精子症患者睾丸内找到精原干细胞，就有可能促使其增殖分化获得精子；或通过特异性标志物分选，体外获得纯度较高的精原干细胞用于增殖培养或移植。然而，如何确定所获得细胞是精原干细胞，成为此项工作的首要条件。精原干细胞在增殖和分化的过程中细胞表面和细胞内会表达许多蛋白质，这些蛋白质如果有较好的特异性和敏感性，作为精原干细胞标志物，将对分离及鉴定精原干细胞发挥重要作用。我们将不同年龄阶段小鼠精原干细胞采用复合酶消化、免疫磁珠分选技术分离纯化出后，通过双向凝胶电泳及基质辅助激光解析电离飞行时间质谱分析系统动态观测各年龄阶段精原干细胞蛋白质表达的差异，运用生物信息学蛋白质组数据库筛选并确定差异性蛋白质或发现新的蛋白质。目前实验已经完成，数据正在整理中，并与深圳华大基因合作进行蛋白质组学分析。