人精子发生相关基因NYD-SP6的功能研究

发布时间：2018-05-14 17:58

本文选题：NYD-SP6 + 精子发生　；参考：《南京医科大学》2006年博士论文

【摘要】：正常的精子发生依赖于生精细胞的分裂分化和生精细胞凋亡之间的平衡，其背后是错综复杂的基因-蛋白调控网络。寻找并掌握这些基因／蛋白的功能，将能更深入地阐明精子发生的分子调控机理。 NYD-SP6是本实验室克隆出的一条编码PHD蛋白的新基因，前期在mRNA水平对其进行的一些初步研究已经高度提示它是一条新的人精子发生相关基因。本研究中，，我们对NYD-SP6基因在精子发生过程中的明确作用及其机制进行了探讨。首先我们在蛋白水平检测了NYD-SP6在人睾丸组织中的细胞定位。构建了pET-22b-NYD-SP6(全长ORF)原核表达质粒，经IPTG诱导得到49KD原核表达蛋白。用纯化的蛋白免疫BalB／C小鼠后获得NYD-SP6多克隆抗体。进一步进行人睾丸组织免疫组化分析，显示NYD-SP6蛋白在包括精原细胞、精母细胞和精子细胞的各级生精细胞核中表达，提示NYD-SP6可能直接调控生精细胞的某种(些)生物学过程，影响精子发生。进一步我们以生精细胞为研究平台，检测NYD-SP6在精子发生过程中的作用。由于NYD-SP6在小鼠中有高度同源的基因，因此我们在小鼠精原细胞系中超表达NYD-SP6，进行了以下功能研究： ① 应用流式细胞术检测了超表达组细胞与对照组细胞的细胞周期，结果显示两组在G_0／G_1期、S期及_G2／M期的细胞比例均无显著差异。提示NYD-SP6对生精细胞的分裂增殖无影响。 ② 应用流式细胞术、DNA ladder电泳、彗星实验三种方法检测了超表达组细胞与对照组细胞分别在自发情况和化学药物Taxol诱导
[Abstract]:Normal spermatogenesis depends on the balance between the division and differentiation of spermatogenic cells and the apoptosis of spermatogenic cells. Finding and mastering the function of these genes / proteins will further clarify the molecular regulation mechanism of spermatogenesis. NYD-SP6 is a novel gene encoding PHD protein cloned in our laboratory. Some preliminary studies on it at the mRNA level have highly indicated that it is a new human spermatogenesis related gene. In this study, we investigated the role and mechanism of NYD-SP6 gene in spermatogenesis. We first examined the cellular localization of NYD-SP6 in human testis at the protein level. The prokaryotic expression plasmid pET-22b-NYD-SP6 (full-length ORF) was constructed and 49KD prokaryotic expression protein was obtained by IPTG induction. NYD-SP6 polyclonal antibody was obtained by immunizing BalB/C mice with purified protein. Further immunohistochemical analysis of human testis showed that NYD-SP6 protein was expressed in spermatogenic nuclei, including spermatogonium, spermatocytes and sperm cells, suggesting that NYD-SP6 may directly regulate some biological process of spermatogenic cells. Affect spermatogenesis. Further, we used spermatogenic cells as a platform to detect the role of NYD-SP6 in spermatogenesis. Since NYD-SP6 has highly homologous genes in mice, we overexpression NYD-SP6 in mouse spermatogonium cell lines. 1 flow cytometry was used to detect the cell cycle between the overexpression group and the control group. The results showed that there was no significant difference between the two groups in the proportion of cells in S phase and G 2 / M phase in G_0/G_1 phase. The results suggest that NYD-SP6 has no effect on the division and proliferation of spermatogenic cells. (2) flow cytometry and comet assay were used to detect the spontaneous and chemical Taxol induction of the cells in the hyper-expression group and the control group, respectively.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R321

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