【作者】 周永翠；

【导师】 秦达念；

【作者基本信息】 汕头大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 目的睾丸的精子发生是一个复杂的细胞分裂分化过程,是一系列基因按时空顺序表达与睾丸组织内外环境共同作用的结果。许多在睾丸中特异或高度表达的基因参与这一过程。本研究在实验室前期工作的基础上,选择三个差异表达的基因TSG23,T279和TSC21进行研究。通过对上述三种基因进行生物信息学,mRNA和蛋白质的表达特性,及在正常和无精症睾丸中的表达差异分析,探讨它们在精子发生过程中的作用,为男性不育症的诊断和治疗提供新的思路和方法。方法(1)应用芯片技术筛选精子发生相关基因。(2)应用生物信息学方法分析TSG23和T279基因和编码蛋白的理化结构。(3)应用PCR方法检测TSG23和T279基因在人和小鼠多组织中及在小鼠不同发育阶段睾丸组织和不同小鼠生殖细胞系中的表达特征;比较它们在正常睾丸及无精症睾丸中的表达差异。(4)制备TSG23、T279和TSC21基因的RNA探针,采用原位杂交的方法检测睾丸组织中mRNA的表达。(5)构建小鼠pEGFP-N1-T279重组质粒载体,转染细胞系,确定T279蛋白的亚细胞定位。(6)构建人pET32a-T279和TSC21表达载体,转化到感受态BL21细菌,诱导表达并纯化T279和TSC21蛋白;免疫小鼠,制备抗人的TSC21多克隆抗体和T279单克隆抗体;应用合成的人多肽抗原免疫小鼠制备TSG23多克隆抗体。(7) Western Blot检测TSG23和T279蛋白和抗体的特异性。(8)免疫荧光方法检测T279在人睾丸组织中的表达。(9)应用免疫组化方法检测TSG23、T279和TSC21蛋白在睾丸组织中的表达定位,并检测它们在正常人睾丸组织和生精阻滞患者睾丸组织中的表达差异。结果(1)表达谱芯片结果显示TSG23和T279基因在成人睾丸中高表达,在小鼠睾丸中于18日龄睾丸中开始表达,且随年龄逐渐升高。(2)人TSG23定位在20q13.12,cDNA全长752bp,编码227个氨基酸。小鼠TSG23定位在2H3,cDNA全长755bp,编码226个氨基酸。人T279定位于13q34,cDNA全长773 bp,编码195个氨基酸。小鼠T279定位于8A2,cDNA长度761bp,编码182个氨基酸。TSG23和T279皆为人—小鼠同源基因。(3) PCR结果显示TSG23和T279为睾丸组织特异性表达。小鼠TSG23和T279仅在出生后15天的睾丸组织中开始表达,且随着年龄的增加逐渐增强,TSG23在Sertoli细胞,精原细胞和精母细胞系中均没有表达,而T279仅在精母细胞系中表达。与正常睾丸组织比较,TSG23和T279基因在无精症睾丸组织的表达减弱或消失。(4)成功制备TSG23、T279和TSC21的正义和反义探针。原位杂交显示人和小鼠睾丸生精上皮的精母细胞和圆形精子细胞内存在TSG23的强阳性杂交信号,并呈现阶段特异性表达模式。小鼠T279mRNA主要定位于靠近管腔的精子细胞及精子中,精母细胞也有阳性信号,TSC21 mRNA定位于小鼠精母细胞和精子细胞的胞浆中。(5)荧光显微镜观察T279融合蛋白定位在精母细胞的内质网中。(6)成功制备人TSG23、T279和TSC21的鼠源性抗体。(7) Western Blot结果显示TSG23蛋白只在睾丸组织中识别出约23KD大小的特异性条带,T279蛋白在睾丸组织中识别出约22KD大小的特异性条带,而在其他组织中两者均为检测到条带。(8)免疫荧光结果表明T279蛋白主要在正常人睾丸组织的精母细胞和圆形精子细胞中表达。(9)免疫组化结果显示在正常睾丸中TSG23、T279和TSC21蛋白主要在精母细胞和圆形精子细胞中表达,且TSG23主要表达于生精过程中的Ⅲ和Ⅴ期,而在Ⅰ和Ⅱ期中表达很低。在生精阻滞的无精症患者中三个蛋白表达均减弱或消失。结论(1) TSG23、T279和TSC21均为睾丸特异性高表达基因,且是人—小鼠同源性基因。在小鼠睾丸组织中的表达呈年龄依赖性升高。(2) TSG23、T279和TSC21在无精子症患者睾丸组织的表达明显减少或消失。(3) TSG23、T279和TSC21基因对于睾丸发育和精子发生可能具有重要作用,其表达降低或消失可能是男性不育症发生的重要原因之一。更多还原

【Abstract】 Objective Spermatogenesis is a highly ordered and complicated process of cellular proliferation and differentiation in the testis. Many genes specifically or highly expressed in the testis are involved in the process of spermatogenesis. According to the previous data in the lab, the present study choose three genes, TSG23, T279 and TSC21, to further investigate the expression characteristics in human and mouse testis, which may provide a new method for the diagnosis and treatment of male infertility and a target for male contraception.Methods (1) Identified and screened the genes related to spermatogenesis by microarray. (2) Analyzed the construction of the genes and its protein by bioinformatics methods. (3) Detected the expression of TSG23 and T279 in human and mouse multi-tissues, in the mouse testis in different stages and in different germ cell lines by PCR; and compared the difference of these genes in normal and azoospermic testis. (4) Detected the localization of TSG23, T279 and TSC21 mRNAs in testis by in situ hybridization. (5) Detected the subcellular localization in mouse cells by the transfection of pEGFP-N1-T279 recombinant plasmid. (6) Generated the anti-T279 monoclonal antibody, and anti-TSC21 and anti-TSG23 polyclonal antibodies. (7) Detected the specific of TSG23 and T279 proteins by Western Blot analysis. (8) Detected the expression of T279 protein in human testis by immunofluorescence. (9) Detected the expression and localization of TSG23, T279 and TSC21 proteins in testis and the difference of expression in testis from fertile men and the patients with azoospermia by immunohistochemistry.Results (1) Affymetrix Genechip analysis showed that TSG23 and T279 were highly expressed in adult human and mouse testis. (2) Human TSG23 gene was mapped to chromosome 20q13.12. The full cDNA length of TSG23 was 752bp, which encoded a protein with 227 amino acids. Mouse TSG23 gene was mapped to chromosome 2H3. The full cDNA length was 755bp, which encoded a protein with 226 amino acids. Human T279 gene was mapped to 13q34. The full cDNA length of was 773bp, which encoded a protein with 195 amino acids. Mouse T279 gene was mapped to 8A2. The full cDNA length was 761bp, which encoded a protein with 227 amino acids. Both TSG23 and T279 are human-mouse homologous genes. (3) RT-PCR analysis from multiple human and mouse tissues indicated TSG23 and T279 mRNA was only expressed in the testis. Mouse TSG23 and T279 was detected after day 15 and gradually increased with the age. There was no expression or less expression of TSG23 and T279 in azoospermic testis compared with normal testis. (4) In situ hybridization revealed that TSG23 mRNA was located in spermatocytes and round spermatids of the seminiferous tubules in human and mouse testis, and the expression could be stage specific. Mouse T279 mRNA was mainly located in spermatids and sperms of the seminiferous tubules in mouse testis. Mouse TSC21 mRNA was located in spermatocytes and spermatids in mouse testis. (5) Immunofluorescent assay revealed T279 fusion protein located in endocytoplasmic reticulum of spermatocytes. (6) Generated successfully anti TSG23, T279 and TSC21 antibody. (7) Western blot analysis demonstrated that TSG23 was mainly expressed in human testis with molecular weight of about 23 KD. And T279 was mainly expressed in human testis with molecular weight of about 23 KD. (8) Immunofluorescence showed that T279 was predominantly located in spermatocytes and round spermatids. (9) Immunohistochemistry showed that TSG23, T279 and TSC21 were predominantly located in spermatocytes and round spermatids. And intense expression of TSG23 was observed in stages III and V, whereas it was lower at stages I and II of spermatogenesis. In the testis from patients with azoospermia, the expression of the three proteins were significantly decreased or disappeared in spermatocytes and round spermatids.Conclusions (1) TSG23, T279 and TSC21 were testis-specific/enriched expressed and human-mouse homologous genes. Moreover, expression of TSG23 mRNA in mouse testis was developmental dependent. (2) In the testis from patients with azoospermia, the expression of TSG23, T279 and TSC21 were significantly decreased or disappeared at mRNA and protein levels. (3) TSG23, T279 and TSC21 may participate in testicular development and spermatogenesis, and their abnormal expression may be one of the important factors in male infertility.更多还原