采用RNA干扰方法特异性降低胚胎干细胞中Nanog的表达

发布时间：2019-04-02 04:47

【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。
[Abstract]:Embryonic stem cells (es) are a group of cells with multidirectional differentiation potential and self-renewal ability, which are isolated from the inner cell mass. The developmental totipotency of embryonic stem cells gives it a wide range of application prospects in clinical practice. The research on the mechanism of embryonic stem cell totipotency is the premise and foundation of its clinical application. Nanog plays an important role in the maintenance of embryonic stem cell totipotency. In order to study the role of Nanog in maintaining totipotency of embryonic stem cells in detail, we used RNA interference method to specifically reduce the expression of Nanog in embryonic stem cells, and observe the changes of J1 cells accompanied by Nanog expression down-regulation. In this experiment, we designed and synthesized four interference fragments targeting Nanog and transfected J1 cells. The effects of each fragment on the expression of Nanog in J1 cells were detected by RT-PCR,Real-Time PCR,Western blot. The results showed that the interference efficiency of Nanog-P1 on Nanog in J1 cells was more than 90%. After the decrease of Nanog expression, the expression of totipotency-related gene utf1 in J1 cells was significantly decreased, while the expression of differentiation-related gene gata6 was significantly increased. These results suggest that Nanog plays an important role in maintaining totipotency of embryonic stem cells.
【作者单位】：中国医学科学院北京协和医学院血液学研究所;
【基金】：国家自然科学基金资助项目(No.30570357)~~
【分类号】：Q78

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