定量分析诱导山羊体细胞重编程过程中端粒酶的表达变化

Quantitative analysis of telomerase reverse transcriptase gene expression in goat reprogramming cells

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。文中重点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。从关中奶山羊胎儿皮肤分离得到的胎儿成纤维细胞 (GEF)，其增殖能力较强，核型正常 (60条XY)，通过转录因子在体外诱导得到山羊重编程细胞。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，结果表明睾丸组织中TERT的表达显著高于上皮组织 (P＜0.01)，在山羊胎儿的其他组织中TERT也有不同程度的表达。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。

Abstract:

Currently, animal somatic cell reprogramming into the induced pluripotent stem cell (iPS) is one of the hottest research target in the field of cell biology. We focused on the analysis of telomerase reverse transcriptase (TERT) gene expression during goat somatic fibroblasts reprogramming, and investigated the relationship between the expression of TERT and the pluripotency of reprogrammed cells. RNA samples of fetal tissues isolated from Guanzhong milk goat fetus, and the induced goat reprogramming cell clones were used to determine the relative expression levels of TERT by the real-time RT-PCR method. Goat embryonic fibroblasts (GEF) collected from the Guanzhong milk goat with normal karyotype were induced by 4 transcription factors to become reprogramming cells. The expression of TERT in reprogramming cells was detected by Real-time RT-PCR. The results showed that the expression of TERT in testis tissue was higher than that in epithelial tissues (P<0.01). The expression level of TERT was higher in AP staining positive cells than that in AP staining negative cells (P<0.01). This result indicated that TERT activity played an important role in cell reprogramming.