人类睾丸cDNA array制备及睾丸发育/精子发生相关基因表达谱系构建

【作者】 沙家豪；

【导师】 周开亚； 王义权；

【作者基本信息】 南京师范大学 ， 动物学， 2003， 博士

【摘要】 哺乳动物的精子发生是一个独特复杂的细胞分裂分化过程。在睾丸曲精小管中的生精细胞经过分裂分化过程，最终生成精子。精子发生过程中主要经历了三个阶段的变化：精原细胞的增殖，精母细胞的减数分裂和精子细胞的变态过程。精子发生是成年睾丸的一个重要功能，许多在睾丸中特异的和／或高度表达的基因参与完成这一过程。 克隆睾丸精子发生相关基因并研究其功能，阐述精子发生过程中减数分裂和分化变态的分子调控机制，对临床男性不育的诊断和治疗以及男性分子节育等有重要的理论意义，也有重大的临床应用价值。 为了发现组织中表达的新基因，许多基于基因差异显示的技术手段，如抑制性差减杂交（SSH），差异显示逆转录PCR（DDRT-PCR）和cDNA array杂交技术得到了应用和改进。与传统的杂交法相比，cDNA array作为一种功能基因克隆和表达图谱研究方法具有大规模、高通量及结果客观性强等优点，已广泛应用于疾病相关、发育相关、组织相关基因的克隆和表达图谱研究。 为了克隆精子发生相关的基因，我们构建了人睾丸cDNA array，并采用了高通量差异杂交策略：从人睾丸cDNA文库中PCR扩增插入片段，将9216个PCR产物点膜构建成人睾丸cDNA array；抽提胚胎睾丸组织和成人睾丸组织的mRNA，³³P标记的dATP逆转录掺入标记成cDNA探针，分别与cDNA array杂交，获得差异表达克隆。杂交信号差异≥两倍以上的克隆为人胚胎睾丸／成年睾丸差异表达的克隆，测序得到克隆的序列并与基因组数据库数据进行比对分析。 杂交结果显示在阳性克隆中，成人睾丸探针与胚胎睾丸探针杂交信号差异大于两倍的克隆有1522个：1091个克隆在成年睾丸高表达， 南京师范大学博士学位论义43个克隆在胚胎睾丸高表达；特异表达于胚胎睾丸的基因其功能可能与睾丸发育相关，而特异表达于成年睾丸的基因可能与睾丸的功能，特别是精子发生和雄激素分。必密切相关。测序和序列分析结果显示 1522个克隆中有 499个独立基因：其中 358条为已知基因，40条全长新基因和101条因T。将358条已知基因按照其功能划分为7类：细胞分裂、细胞信号传导、细胞结构／运动、机体防御、蛋白表达、代谢和未知类。以上述结果为基础，我们构建了人类睾丸发育／精子发生相关基因表达谱系，在此谱系中很多基因与蛋白表达功能相关。 为了验证array差异杂交的结果，我们对随机选择的array杂交显示在成年睾丸高表达的8条已知基因采用荧光TaqMan实时定量PCR技术进行了基因在人胚胎睾丸／成人事九中表达的实时定量研究，结果显示8条基因在成人睾丸中的表达／胚胎睾丸中的表达均大于3倍以上，证实了array差异杂交结果的正确性。 此外，本研究还用小鼠一周龄和四周龄睾丸组织 CDNA探针在相同的反应条件下分别与人事丸cDNA array进行杂交，结果显示小鼠睾丸组织探针杂交阳性率约为引％，提示人和小鼠睾丸表达的基因有很高的同源性。在杂交的阳性克隆中，四周龄／一周龄小鼠睾丸探针杂交信号差异大于 3倍的克隆有 256个：201个克隆在四周龄。J、鼠睾丸高表达，55个克隆在一周龄，J、鼠睾丸高表达。从上述人和。J、鼠睾丸探针分别杂交 aray的结果中发现有 101个独立基因在人和小鼠睾丸的不同发育阶段差异表达均大于3倍以上，这种在人和小鼠睾丸发育不同阶段差异表达的保守基因可能在睾丸发育和精子发生过程中起重要的作用。在这lin个克隆中，有54条已知基因，5条假设蛋白（oAA）基因，u条全长新基因和引条表达序列标签（ESn）。人和小鼠睾丸同源的54条已知基因按功能同上划分为7类。上述实验结果显示保守的人和小鼠同源基因与睾丸发育和精子发生密切相 2 南京师范大学博士学位论文关，这些基因为应用小鼠动物模型阐明其在人类的睾丸发育和精子发生过程中的作用奠定基础。 综上所述，我们通过构建人睾丸cDNA array并用人和小鼠不同发育阶段的睾丸探针与其进行差异杂交，克隆了40条睾丸发育／精子发生相关的全新基因；发现了101个在人和小鼠睾丸不同发育阶段差异表达的同源保守基因；获得人睾丸差异表达的358条已知基因及人和小鼠同源的54条基因并进行了功能分类，构建了人类睾丸发育／精子发生相关的已知基因表达语系及人和小鼠同源已知基因表达谱系，为后基因组时代精子发生相关基因的功能研究（新基因的功能，已知基因与睾丸发育／精子发生相关的新功能），为进一步应用于男性生育相关药物机理分析及新药筛选、等九相关疾病基因诊断芯片的制备以及男性不育的基因治疗打下了一个坚实的理论基础。更多还原

【Abstract】 Spermatogenesis, the fundamental function of adult testis, is a continuum of cellular differentiation in which three principal phases can be discerned: spermatogonia renewal and proliferation, meiosis and spermiogenesis. It is a complex process involving cell division, differentiation and interactions between cells in the microenvironment of the seminiferous tubule. Many specific genes and/or highly expressed in testis tissue are involved in the process of Spermatogenesis.The cloning of genes related Spermatogenesis and the study of their molecular regulating mechanism are very important for clinical diagnosis and treatment of male infertility. It is also important to male molecular contraception.In order to isolate genes differentially expressed in tissues, a variety of approaches for different display measurement have been developed and improved, such as suppressed subtractive hybridization (SSH), differential display reversed transcripted PCR (DDRT-PCR) and cDNA microarray technology. Compared with traditional hybridizing techniques, cDNA microarray has many advantages, such as its large scale, high throughout, high efficiency and its objectivity. It can be used as a genome-wide approach to functional characterization of large number of genes and their expression profiles. Advancement in technology of microarray construction and experimental strategy has resulted in expansion of many disease-associated, development-associated and special tissue-associated gene cloning and expression profile analysis.In order to clone genes associated with Spermatogenesis, we originally constructed cDNA microarray from the human testis large insert cDNA library. Insert cDNA was amplified from cDNA library and 9,216 individual clones were spotted on a nylon membrane. 8housekeeping genes and 2 plasmid DNAs were used as controls. Then mRNA was extracted respectively from fetus and adult testis, following reverse transcripted to cDNA probe incorporated with 33P-label dATP. The human testis cDNA microarray was hybridized with two probes respectively and the differently expressed clones were obtained. Those clones whose intensity was at least twofold difference were considered as differentially expressed genes in adult or fetal testis. The insert cDNA sequence was obtained by sequencing technology and BLAST with the gene database of GenBank.Among the positive cDNA clones that gave signals, 1522 clones had intensities at least twofold difference between fetus testis and adult testis: 1091 clones had intensities at least twofold higher for probe prepared from adult tissue than those from fetal tissue. Whereas 431 clones had at least twofold higher intensities for probe prepared from the fetal testis tissue than those from adult testis tissue. Those specifically expressed in fetal testis may be related to the development of human testis, whereas those specifically expressed in adult testis might be related to spermatogenesis. Among 1522 clones, sequencing and blast analysis identified 499 unique genes: 358 were found with their sequences reported before, 40 genes were new full length, remaining were 101 ESTs. To establish a functional profile of the 358 reported genes, proteins encoded by these genes were grouped into the following seven broad categories of biological roles: cell division, cell communication, cell structure/motility, organism defense, protein expression, metabolism and unclassified. Based on above results, we constructed expression map of genes related to the development of human testis and spermatogenesis. In this map, most genes were related to protein expression. To confirm above gene differential expression result from microarray hybridization, we relied on the real time PCR technique-TagMan assay to quantitateexpression of some genes in the fetus testis and adult testis respectively. 8 known genes were picked out and microarray hybridizing showed their highly expression in adult testis. Consistent with the microarray data, we measured at least a 3-fold higher level of mRNA for all eig更多还原