人精子发生相关基因NYD-SP6的功能研究

【作者】 祝辉；

【导师】 沙家豪； 周作民；

【作者基本信息】 南京医科大学 ， 组织胚胎学， 2006， 博士

【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G₀／G₁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诱导更多还原

【Abstract】 Normal spermatogenesis is highly dependent on well-balanced germ cell proliferation, differentiation, and apoptosis. These processes are governed by expression of a series genes. Cloning of these genes and further functional study are very important to the elucidation of molecular mechanism involving in spermiogenesis.NYD-SP6 was cloned from human testis by differential cDNA microarray hybridization in our laboratory and predicted to edcode a PHD finger protein. Preliminary studies on mRNA level have predicted that NYD-SP6 is a spermatogenesis related gene. In this study, we clarified the exact role and mechanism of NYD-SP6 in the process of spermatogenesis.Firstly, we examined the localization of NYD-SP6 protein in adult testis. Open reading fragment of NYD-SP6 was cloned into the pET-22b expression vector. The recombinant protein expression was induced with isopropyl-1-thio-b-D-galactopyranoside. The purified recombinant NYD-SP6-His protein was used as an immunogen and injected into BalB/C mice for polyclonal antibody development. Further immunochemical analysis demonstrated that NYD-SP6 protein was expressed in the nucleus of spermatogonia, spermatocyte and spermatid, which implied NYD-SP6 regulated biological process of spermatogenesis directly.Secondly, functional study about NYD-SP6 in the process of spermatogenesis was carried out on the platform of spermatogenic cell. Because NYD-SP6 has highly homologous gene in the mouse,更多还原