[理学]构建EGFP-PDX-1融合表达载体电穿孔转染大鼠胎肝干细胞的研究 _百替生物.pdfVIP

构建EGFP-PDX-1融合表达载体电穿孔转染大鼠胎肝干细 胞的研究 作者：孙冰 ；孙晓艳 ；安靓1 2 1 ( 南方医科大学组织胚胎学1 教研室，广东 广州 510515； 解放军总医院基础医学研究所，北2 京 100853) 摘要：目的 构建PDX-1绿色荧光蛋白融合表达载体，电穿孔转染入大鼠胎肝干细胞 以得到稳定表达。方法 从SK900/ BLSCRIPT质粒中扩增PDX-1，将其插入pEGFP-C1 的多克隆位点中，构建重组质粒pEGFP-C1-PDX-1；分离培养大鼠胎肝干细胞，经鉴 定后用电穿孔法转染；分析转染前后细胞生长曲线，荧光显微镜下观察、RT-PCR鉴 定转染结果。 结果 重组质粒经酶切鉴定正确无误，经电穿孔转染后GFP和PDX-1 基因均能在胎肝干细胞中保持较长时间稳定表达，并对胎肝干细胞的生长增殖影响较 小。结论 成功构建了PDX-1绿色荧光蛋白融合表达载体，能在胎肝干细胞中较稳定 表达，为研究PDX-1在干细胞定向分化为胰岛素分泌细胞中的调控作用提供了物质基 础。 关键词：肝干细胞；绿色荧光蛋白；载体构建；胰十二指肠同源盒基因-1；电穿孔 中图分类号：R321 文献标识码：A 文章编号：1673-4254(2006)06-0750-04 ConstructionoffusionexpressionvectorEGFP-PDX-1anditstransfectioninto rat fetal hepatic stem cells by electroporation SUN Bing； SUN Xiao-yan； AN Jing1 2 1 1Department of Histology and Embryology, Southern Medical University, 2 Guangzhou 510515, China; Institute of Basic Medical Sciences, General Hospital of PLA, Beijing 100853, China Abstract: Objective To construct the fusion expression vector of service@100 service@100 wwwwww..110000bbiiootteecchh..ccoomm sseerrvviiccee@@110000bbiiootteecchh..ccoomm pancreatic-duodenal homeobox gene 1 (PDX-1) fused to green fluorescent protein (GFP) capable of stable expression in fetal rat hepatic stem cells aftertranfectionbyelectroporation.MethodsPDX-1cDNAwasamplified from SK900/BLSCRIPT plasmid and cloned into the multiple cloning site of pEGFP-C1 to obtain the recombined plasmid pEGFP-C1-PDX-1. Rat fetal hepatic stem cells were isolated, cultured, identified and transfected with the recombinantvectorbyelectroporation,followedbyobservationofthesecells withfluorescentmicroscope.TheresultoftransfectionwasanalyzedbyRT-PCR