【摘要】 目的:通过在人胚胎干细胞(hESC)中有效转染微小RNA miR-125b的真核表达载体,研究过表达miR-125b对hESC增殖的影响。方法:将在无饲养层上培养至第3 d,克隆融合达70%的hESC用Accutase酶消化为单细胞,然后用LipofectAMINE2000对hESC单细胞转染pHRS-1cla-miR125b-CMV-EGFP载体及其对照pHRS-1cla-CMV-EGFP载体,通过实时定量PCR对转染后细胞中成熟miR-125b的表达进行检测;进一步进行细胞计数和克隆计数,对miR-125b表达上调的hESC的增殖情况进行分析。结果:实时定量PCR检测结果表明,细胞转染后72 h,miR-125b的表达上调1.45倍,说明hESC转染成功;克隆计数及细胞计数结果显示过表达miR-125b的hESC增殖受到明显抑制(P<001)。结论:转染miR-125b真核表达载体的hESC能够上调成熟miR-125b的表达,hESC中miR-125b的表达上调能明显抑制hESC的增殖。更多还原

【Abstract】 Objective: To study the effect on human embryonic stem cells(hESC) proliferation when overexpress ing microRNA 125b(miR-125b) through transfecting human embryonic stem cells with eukaryotic expression vector of miR-125b.Methods: Dissociated the hESC clones into single cells which cultured on matrigel for 3 d and about 70% confluence.After the experimental group plasmid pHRS-1cla-miR125b-CMV-EGFP and the control group pHRS-1cla-CMV-EGFP plasmid transfection with LipofectAMINE2000,detected the expression of miR-125b with realtime PCR,then counted the single cell number and clone number and analysis the regulation effect fur ther.Results: The realtime PCR result after transfecting 72 h showed that the expression of miR-125b had a up-regulation,and suggest that we had done an effective transfection.The single cell number and clone number in dicated that miR-125b suppressed the proliferation of hESC significantly(P<001).Conclusion: The realtime PCR result suggest that after transfecting eukaryotic expression vector pHRS-1cla-miR125b-CMV,the mature miR-125b had a significant up-regulation in hESC,and the up-regulation of miR-125b inhibit hESC proliferation obviously.更多还原