三个新的精子发生相关基因在人和小鼠睾丸中的表达特征

发布时间：2018-09-05 19:34

【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。
[Abstract]:Objective Testicular spermatogenesis is a complex process of cell division and differentiation, which is the result of a series of genes expressed in time-space sequence and interaction with the internal and external environment of testicular tissue. The three genes TSG23, T279 and TSC21 were studied. Their bioinformatics, expression characteristics of mRNA and protein, and differential expression in normal and azoospermic testis were analyzed to explore their roles in spermatogenesis, and to provide new ideas and methods for the diagnosis and treatment of male infertility.
Methods (1) Screening of spermatogenesis related genes by microarray technique. (2) Analyzing the physical and chemical structure of TSG2 3 and T279 genes and coding proteins by bioinformatics method. (3) Detecting the expression of TSG2 3 and T279 genes in human and mouse multiple tissues, testicular tissues at different developmental stages and different mouse germ cell lines by PCR method. The expression of TSG23, T279 and TSC21 was detected by in situ hybridization. (5) The recombinant plasmid vector of mouse pEGFP-N1-T279 was constructed and transfected into cell lines to determine the subcellular localization of T279 protein. (6) Human pET32a-T279 was constructed. TSC21 and TSC21 expression vectors were transformed into competent BL21 bacteria to induce the expression and purification of T279 and TSC21 proteins; mice were immunized with polyclonal antibodies against human TSC21 and monoclonal antibodies against T279; mice were immunized with synthetic human polypeptide antigens to prepare TSG23 polyclonal antibodies. (7) Western Blot was used to detect the specificity of TSG23 and T279 proteins and antibodies. (8) Immunization. The expression of TSG23, T279 and TSC21 in human testicular tissue was detected by fluorescence staining. (9) The expression and localization of TSG23, T279 and TSC21 proteins in testicular tissue were detected by immunohistochemical method, and the expression differences of TSG23, T279 and TSC21 proteins in normal human testicular tissue and testicular tissue of patients with spermatogenesis block were detected.
Results (1) TSG23 and T279 genes were highly expressed in adult testis, and began to express in 18-day-old testis of mice, and gradually increased with age. (2) Human TSG23 was localized at 20q13.12, with a full length of 752 bp, encoding 227 amino acids. TSG2 3 and T279 were homologous to human-mouse genes. (3) PCR results showed that TSG2 3 and T279 were specifically expressed in testicular tissues. TSG2 3 and T279 were expressed only in testicular tissues 15 days after birth and began to express with the passage of the year. The expression of TSG23 and T279 in spermatocyte, spermatogonia and spermatocyte was decreased or disappeared compared with normal testicular tissues. (4) Sensitive and antisense probes for TSG23, T279 and TSC21 were successfully prepared. The results showed that there were strong positive hybridization signals of TSG23 in spermatocytes and round sperm cells of human and mouse testicular seminiferous epithelium, and the expression pattern was stage-specific. In cytoplasm. (5) The fusion protein T279 was localized in the endoplasmic reticulum of spermatocytes by fluorescence microscopy. (6) Mouse-derived antibodies against human TSG23, T279 and TSC21 were successfully prepared. (7) Western Blot results showed that the TSG23 protein only recognized a specific band of about 23 KD in testicular tissue, and the T279 protein recognized a specific band of about 22 KD in testicular tissue. Specific bands were detected in other tissues. (8) Immunofluorescence showed that T279 protein was mainly expressed in spermatocytes and round sperm cells of normal human testis. (9) Immunohistochemistry showed that TSG23, T279 and TSC21 proteins were mainly expressed in spermatocytes and round sperm cells of normal testis. TSG23 was mainly expressed in stages III and V of spermatogenesis, but very low in stages I and II.
Conclusion (1) TSG23, T279 and TSC21 are testicular-specific high-expression genes, and are homologous to human-mouse genes. The expression of TSG23, T279 and TSC21 in testicular tissues of mice increases in age-dependent manner. (2) The expression of TSG23, T279 and TSC21 in testicular tissues of azoospermia patients decreases or disappears significantly. (3) The expression of TSG23, T279 and TSC21 genes in testicular development and sperm disappears. It may play an important role in the occurrence of male infertility, and the decrease or disappearance of its expression may be one of the important reasons for the occurrence of male infertility.
【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R321

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