采用RNA干扰方法特异性降低胚胎干细胞中Nanog的表达  

Down-regulate Nanog Expression in Embryonic Stem Cells Using RNA Interference

作　　者：田雪梅[1] 刘娜[1] 陆敏[1] 

机构地区：[1]中国医学科学院北京协和医学院血液学研究所,天津300020

出　　处：《细胞生物学杂志》2009年第1期113-118,共6页Chinese Journal of Cell Biology

基　　金：国家自然科学基金资助项目(No.30570357)~~

摘　　要：胚胎干细胞是从内细胞团分离得到的一群具有多向分化潜能和自我更新能力的细胞,发育上的全能性赋予其在临床上广泛的应用前景。对胚胎干细胞全能性机制的研究是其在临床上得以应用的前提和基础。Nanog在胚胎干细胞全能性的维持中起着至关重要的作用,为了更详尽地研究Nanog在胚胎干细胞全能性维持中的作用,我们采用RNA干扰的方法,特异性地降低胚胎干细胞中Nanog的表达,并观察伴随Nanog的表达下降J1细胞的变化。在本试验中,我们设计合成了四条针对Nanog的干扰片段,转染J1细胞,利用RT-PCR、Real-Time PCR、Western印迹检测各个片段对J1细胞中Nanog表达的影响。结果显示,Nanog-P1对J1细胞中Nanog的干扰效率可达90%以上。Nanog表达下降后J1细胞中全能性相关基因utf1的表达显著降低,而分化相关基因gata6的表达有明显升高,说明Nanog在胚胎干细胞的全能性维持中起重要作用。Embryonic stem ceils are derived from inner cell mass of blastocyst. Embryonic stem ceils have two defining properties, self-renewal and pluripotency, which make them very attractive in clinic. Understanding of the mechanism involved in the pluripotency is essential for achieving these goals. Nanog plays a crucial role in the maintenance of pluripotency. In order to investigate Nanog mechanisms in embryonic stem cells pluripotency, we used RNA interference to specifically down-regulate Nanog expression in embryonic stem cells and investigated the change of J1 cells following with Nanog down-regulation. Here, we designed four siRNAs on the basis of Nanog sequence. Then we analyzed Nanog expression using RT-PCR, Real-Time PCR, and Western blot after these siRNAs were transfected in J1 cells. We observed that Nanog-P1 significantly down-regulated Nanog expression in J1 cells, the precise interference rate of Nanog-P1 was up to 90%. Following Nanog expression was silenced, utfl （a pluripotency marker of ES cells） expression was decreased but gata6 （a differentiation related gene） was up- regulated. Our experiments showed that Nanog played an important role in ES cells pluripotency.

关 键 词：胚胎干细胞 NANOG RNA干扰 全能性 

分 类 号：Q78[生物学—分子生物学]