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目的:建立胚胎生殖细胞中沉默多能干细胞特异转录因子Nanog表达的方法。方法:通过检测碱性磷酸酶、SSEA-1和Oct4标志蛋白,鉴定体外培养的胚胎生殖细胞(EG细胞),通过脂质体介导作用,将Nanog特异性小干扰RNA(siRNA)转染EG细胞,应用半定量RT-PCR检测转染24h、36h、48h后EG细胞中Nanog mRNA的表达水平,并应用免疫荧光技术检测转染36h、48h后EG细胞中Nanog蛋白的表达水平。结果:培养细胞具有高度碱性磷酸酶活性,SSEA-1、Oct4阳性,为保持未分化状态的EG细胞。转染后Nanog mRNA和蛋白表达均受到抑制。结论:脂质体介导siRNA能有效沉默EG细胞中Nanog基因的表达,为进一步探讨Nanog对胚胎生殖细胞的调控机制奠定了基础。
OBJECTIVE: To establish a method for Nanog expression of silencing pluripotent stem cell-specific transcription factor in embryonic germ cells. Methods: Embryonic germ cells (EG cells) were identified by detecting alkaline phosphatase, SSEA-1 and Oct4 marker proteins. Nanog-specific small interfering RNA (siRNA) was transfected into EG The expression of Nanog mRNA in EG cells was detected by semi-quantitative RT-PCR at 24h, 36h, 48h after transfection. The expression of Nanog protein in EG cells was detected by immunofluorescence technique at 36h and 48h after transfection. Results: The cultured cells were highly alkaline phosphatase activity, SSEA-1, Oct4-positive, to maintain the undifferentiated state of EG cells. After transfection Nanog mRNA and protein expression were inhibited. CONCLUSION: Liposome-mediated siRNA can effectively silence the expression of Nanog gene in EG cells, which lays the foundation for further exploration of the regulatory mechanism of Nanog on embryonic germ cells.