两种传代方法影响人胚胎干细胞转染效率的比较

背景:现有方法将外源分子如DNA导入到人胚胎干细胞用于科学研究的效率普遍较低,如何优化现有条件,提高转染效率显得尤为重要。目的:比较两种不同的传代方法对人胚胎干细胞系H9转染效率的影响,优化胚胎干细胞转染条件。方法:人胚胎干细胞系H9分别采用小克隆传代法和单细胞传代法进行传代,传代后继续培养细胞48 h,用Lipofectamine 3000转染pAdTrack-AKT1荧光质粒2 d后,荧光显微镜下观察荧光质粒的表达,流式细胞仪检测人胚胎干细胞的转染效率;RT-qPCR和Western blot分别检测转染后AKT1在mRNA和蛋白质水平的表达。结果与结论:①荧光显微镜下观察发现单细胞传代组表达荧光质粒的细胞数量更多,流式细胞仪检测单细胞传代法的转染效率(47.18±2.00)%]高于小克隆传代法的转染效率(19.52±0.86)%],差异有显著性意义(P<0.01);②单细胞传代组转染后AKT1 mRNA和蛋白的表达均高于小克隆传代组,差异有显著性意义(P<0.01);③结果表明,采用单细胞传代法,增加细胞与转染试剂脂质体的接触面积可提高人胚胎干细胞的转染效率。

Comparison of two passage methods affecting the transfection efficiency of human embryonic stem cells

BACKGROUND:In the research of human embryonic stem cells,introducing exogenous molecules such as DNA into cells is a common research method,but the transfection efficiency is relatively low.It is crucial to answer the question of how to optimize the existing conditions to improve the transfection efficiency.OBJECTIVE:To compare the effects of two different passaging methods on H9 transfection efficiency,in order to optimize the conditions required for embryonic stem cell transfection.METHODS:Human embryonic stem cell lines H9 were cultured for 48 hours after small clone passaging or single-cell passaging.Lipofectamine 3000 was used to transfect pAdTrack-AKT1 fluorescent plasmid into human embryonic stem cells.After 2 days of transfection,the expression of fluorescent plasmids was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry.RT-qPCR and western blot were used to detect the mRNA and protein expression levels of AKT1 respectively.RESULTS AND CONCLUSION:Under the fluorescence microscopy,the number of cells expressing fluorescent plasmids in the single-cell passaging group was more than that in the small clone passaging group,and the flow cytometry analysis showed that the transfection efficiency of cells in the single-cell passaging group was(47.18±2.00)%,which was significantly higher than(19.52±0.86)%in the small clone passaging group(P<0.01).RT-qPCR and western blot analysis showed that the expression levels of AKT1 mRNA and protein in the single-cell passaging group were significantly higher than those in the small clone passaging group(P<0.01).These findings indicate that single-cell passaging can increase the contact area between cells and transfection reagent liposomes,and improve the transfection efficiency of human embryonic stem cells.