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自2006年诱导多能干细胞(iPS)技术诞生以来,采用病毒等载体进行的诱导方法已取得了成功.但是,其致瘤性的影响限制了病毒载体的推广与应用,而采用非病毒载体诱导iPS细胞成为研究的热点.本研究通过2个启动子的独立启动,构建了带有绿色荧光标记的OCT 4/SOX 2共表达诱导载体(pOct4/Sox2-EGFP).将该载体转染HEK 293FT细胞后,阳性克隆明显表达绿色荧光.并通过RT-PCR、免疫荧光等方法证明其中的转录因子OCT 4和SOX 2能在转染细胞中高效表达,同时诱导受体细胞中内源NANOG的转录表达.本研究说明,OCT 4/SOX 2共表达载体能激活NANOG基因的内源表达,暗示着非病毒不整合载体pOct4/Sox2-EGFP本身或与其它转录因子和小分子结合可用于诱导成体细胞的重编程.因此,本研究为下一步应用质粒载体诱导体细胞重编程为iPS细胞的研究奠定了工作基础.
Induction methods such as virus vectors have been successful since the birth of induced pluripotent stem cell (iPS) technology in 2006. However, their tumorigenicity has limited the promotion and application of viral vectors, but has been induced by non-viral vectors iPS cells have become the focus of research.In this study, we constructed an OCT 4 / SOX 2 co-expression vector (pOct4 / Sox2-EGFP) with green fluorescent labeling through the independent promoter of two promoters.The vector was transfected into HEK 293FT The positive clones expressed green fluorescence obviously, and the transcription factors OCT 4 and SOX 2 were proved to be highly expressed in the transfected cells by RT-PCR and immunofluorescence, and the transcription of endogenous NANOG was also induced in the recipient cells Expression.This study shows that OCT4 / SOX2 co-expression vector can activate the endogenous expression of NANOG gene, suggesting that the non-viral non-integrating vector pOct4 / Sox2-EGFP itself or in combination with other transcription factors and small molecules can be used to induce adult cells Therefore, this study laid the foundation for the next step of using plasmid vector to induce somatic cell reprogramming into iPS cells.