【作者】 张淑金；

【导师】 王华岩；

【作者基本信息】 西北农林科技大学 ， 动物生物技术， 2010， 硕士

【摘要】 动物体细胞重编程为诱导多能干细胞(iPS)是目前干细胞生物学研究的热点。在人、猴、大鼠、猪上都得到了iPS细胞,说明iPS技术可以广泛应用于建立动物iPS细胞系。本文从关中奶山羊胎儿皮肤分离得到的胎儿成纤维细胞(GEF),通过4个转录因子在体外诱导得到山羊重编程细胞。并对诱导的山羊重编程细胞进行了一系列的鉴定和检测。利用Real-time RT-PCR方法首先对关中奶山羊胎儿各种组织的TERT表达进行了检测,然后重点是对山羊体细胞重编程过程中端粒酶(TERT)基因的相对表达量进行了检测,探讨了山羊重编程细胞的形成与端粒酶基因表达的关系。得出的结论主要有以下几点:1.采取关中奶山羊胎儿皮肤,利用组织块法和胰酶消化法分离培养得到了胎儿成纤维细胞(GEF)。其增殖能力较强,生长旺盛,核型正常(60条XY)。GEF作为诱导山羊iPS的受体细胞,由生长曲线可以看出传代次数越少越好。所以实验中选用前3代的GEF细胞。2.利用Real-time RT-PCR方法测定关中奶山羊胎儿八种组织端粒酶(TERT)基因的相对表达量,组织中TERT基因也有不同程度的表达。结果表明睾丸组织中TERT的表达显著高于其他组织(P<0.01)。3.诱导山羊体细胞重编程是个缓慢的过程,一般需要15-20 d以上的培养时间。诱导的细胞在第10-12 d开始出现克隆集落,周围的细胞逐渐开始聚团,从成纤维细胞时的长梭样形态变为圆形并聚集成集落。初始的克隆集落较小,挑单克隆之后可以继续传代培养,且保持基本的细胞形态。持续培养15d后,克隆集落长大,成圆形或者椭圆形,边缘整齐,核质比较高,挑克隆,进行传代培养。4.诱导山羊重编程细胞AP染色阳性克隆很少。AP阳性的克隆Oct4、Nanog、Tert免疫荧光阳性,Tert免疫组化阳性。说明重编程细胞表达内源性基因,表达端粒酶。5.利用Real-time RT-PCR方法对原代重编程细胞和4株不完全重编程细胞株的TERT表达检测结果发现,碱性磷酸酶(AP)阳性的重编程细胞端粒酶表达量要显著高于AP阴性的重编程细胞(P<0.01)。这一结果揭示,激活端粒酶活性并使其保持较高的表达水平对体细胞的重编程至关重要。更多还原

【Abstract】 Animal somatic cells can be reprogrammed to induced pluripotent stem cells (iPS) that has recently become a hot research area. iPS cells have been generated from human, monkey, rat and pig, indicating that this technology could be widely used in establishing animal iPS cell lines. In this study, the goat embryonic fibroblasts (GEF) that was isolated from Guanzhong milk goat fetus were induced to reprogrammed cells (iPS) by four transcription factors in vitro. Based on Real-time RT-PCR method, we determined the expression of TERT in different tissues of Guanzhong milk goat fetus, gost fibroblasts and pips. We also conferred the relationship between goat reprogrammed cell formation and telomerase gene expression level. The main conclusions are as follows:1. The goat embryonic fibroblasts (GEF) from skin of Guanzhong milk goat fetus were isolated by using both the tissue culture and enzyme digestion methods. The isolated GEF had the good proliferation capability and the normal karyotype (60 XY). We chose the early passage (2-5) GEF as the recipient cells for the iPS induction.2. The relative expression level of eight organizations’telomerase (TERT) gene in Guanzhong dairy goat embryo was determined by using Real-time RT-PCR; and there had also varying degrees of TERT gene expression in other organizations. The results showed that the expression of TERT in testis was significantly higher than that of the epithelial tissue (p <0.01).3. Goat somatic cell reprogramming is a slow process, it normally takes more than 15-20d. Induced cells began to become a clone in 10-12d,and the cells around gradually began to gather,and finally formed a round colony which followed with a fibroblast-like shape to a round shape.The initial colony was small,and it can be continuously cultured after picking up the monoclonal, and it also can maintain the basic morphology.After 15d continuous culturation, the clone grew bigger, into a round or oval shape,and the edge of which is neat, the karyoplasmic ratio is relatively high, and then pick clones again and culture them continuously.4. AP stain positive clones in goat induced reprogramming cells were rarely. AP-positive clones were also positive in Oct4/Nanog/Tert immunofluorescence and Tert immunohistochemical staining, indicating that the reprogramming cells expressed endogenous gene and high telomerase activity.5. Using Real-time RT-PCR method to test the expression of TERT between reprogrammed primary cells and four incomplete reprogrammed cell lines, the results showed that the telomerase expression in alkaline phosphatase(AP)-positive reprogrammed cells was significantly higher than in AP-negative cells (p <0.01).更多还原