共表达外源OCT4/SOX2可激活人胚肾细胞中内源NANOG的表达

Co-expression of OCT4/SOX2 Activates Endogenous NANOG Expression in Human Embryonic Kidney Cells

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.

Induced pluripotent stem cell（iPS） was first reported through ectopic expression of defined factors from somatic cells in 2006.The iPS cells derived from patients are extremely useful for drug discovery and regenerative medicine.To avoid the risk of tumorigenicity of viral-mediated iPS technology that limited its application in both developmental and clinical research,we construct an OCT 4/SOX 2 co-expression vector（pOct4/Sox2-EGFP） controlled by two promoters and contained an EGFP marker.By transfecting pOct4/Sox2-EGFP vector into HEK 293FT cells,the positive clones with green fluorescence were screened for the express of exogenous OCT 4 and SOX 2 by immunocyto-chemistry.At 48 hours after transfection,the transcription of NANOG gene was detected,which was absent in control HEK 293FT cells.The results indicated that co-expression of OCT 4/SOX 2 can activate the expression of endogenous NANOG gene,and suggesting that the pOct4/Sox2-EGFP might be an useful non-viral vector for iPS cells studies.