【作者】 向阳；

【导师】 卢光琇；

【作者基本信息】 中南大学 ， 遗传学， 2006， 博士

【摘要】 研究目的：对核型正常和异常的人类胚胎干细胞(hESCs)在体外培养过程中Wnt基因在饲养层和hESCs中的差异表达分析，分别筛选出可能促进hESCs正常增殖和引起核型发生变化的候选Wnt基因，并对其中的Wntga候选基因进行RNAi(RNA干扰)研究，了解其生理功能；在小鼠睾丸或生殖域中克隆与生精相关的新基因，并对其功能进行初步研究。 研究方法：用RT-PCR方法对核型正常和异常的hESCs在体外培养过程中Wnt基因在饲养层和hES细胞中的差异表达分析，分别筛选维持hESCs正常增殖和促使其核型变化的候选基因：用RT-PCR、原位杂交、免疫组化、细胞定位和过表达方法研究其定位和表达，并构建pAVU6+27／siWnt9as针对人Wnt9a RNA干扰的表达载体，采用原位杂交方法检测RNA干扰效应，以及流式分析过表达和低表达人Wnt9a对细胞周期的影响；应用DDD(数据库削减杂交)结合实验方法，克隆在睾丸或生殖域中特异表达的新基因，采用RT-PCR、Northern、原位杂交、免疫组化、蛋白表达和细胞定位等方法研究新基因的初步功能。 研究结果：①Wnt7a在培养核型正常hESCs 6天后的hEFs(人类胚胎成纤维细胞)和ICR小鼠胚胎成纤维细胞(mEFs)中表达，在对照和培养第一号染色体核型异常的hESCs 6天后的hEFs和ICR mEFs中无表达。Wnt3a在培养核型正常hESCs 6天后的ICR mEFs中高表达，在对照和培养核型异常hESCs 6天后的ICR小鼠mEFs中无表达。Wnt3在培养第一号染色体核型异常的hESCs 6天后的hEFs和ICRmEFs中表达，且表达量逐渐增强，对照和培养核型正常hESCs后的hEFs中无表达；但培养正常hESCs 6天后的ICR和昆明小白鼠mEFs中有Wnt3的表达；同时，昆明小白鼠mEFs对照中也有Wnt3的表达。Wnt9a的表达量在未培养正常hESCs的hEFs中表达量高，而在养hESCs后的hEFs中表达量低。Wnt16在培养正常hESCs 6天后的昆明小白鼠mEFs中有较高的表达，而对照和hEFs及ICR小鼠mEFs中均无Wnt16表达。②Wnt9a在人3个月胚胎的多组织中均表达；原位杂交和免疫组化表明Wnt9a在MCF-7人乳腺癌细胞和hESCs中表达；Wnt9a定位在胞浆中表达；原位杂交检测表明转染了pAVU6+27/siWnt9as的MCF-7细胞无杂交信号或杂交信号弱，未转染更多还原

【Abstract】 Objective: The aims of our research are ① to filter candidate Wnt genes which are important for maintenance of normal hESCs or resulting in karyotypic change through analysis of Wnt genes of differential expression in human and mouse feeder cells and hESCs during culture of normal or abnormal karyotype hESCs. ② to research the function of Wnt9a that RNAi for human Wnt9a were performed. ③ to clone the spermatogenesis-related new genes expressed in mouse testes or reproductive tract and to study their function.Methods: ① differential expression analyses of Wnt genes in human and mouse feeder cells and hESCs during culture normal or abnormal karyotype hESCs were carried out to filter candidate Wnt genes which are important for maintenance of the normal hESCs or resulting in karyotypic change. Analyses of RT-PCR, in situ hybridization, immunohistochemistry and localization were performed to study the function of human Wnt9a. ② By synthesizing siRNAs, the pAVU6+27/siWnt9as expression vectors were constructed for human Wnt9a RNAi and then use in situ hybridization was performed to examine the effect of siRNAs for Wnt9a. MTT assay were carried out to analyze cell cycle and apoptosis at over expression and lower expression Wnt9a in MCF-7 cells. ③ DDD and experiment methods were performed to clone new genes expressed in mouse testis or reproductive tract and to study their functions by RT-PCR, northern blotting, in situ hybridization, Immunohistochemistry, protein expression and cell location analysis.Results: ① Wnt7a was expressed in hEFCs and ICR mEFCs after culture normal karyotype hESCs, but not expressed in control and in hEFCs and ICR mEFCs after culturing abnormal karyotype hESCs with karyotypic change in chromosome NO. 1. Wnt3a was expressed in ICR and KUNMINGBAI mouse mEFCs after culturing normal karyotype hESCs, but not expressed in control and in mEFCs after culture abnormal karyotype hESCs. Wnt3 was expressed in ICR mEFCs and hEFCs after culture abnormal karyotype hESCs, not expressed in control, but更多还原