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.doc

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1;孙晓艳2;安靓1 (1南方医科大学组织胚胎学教研室,广东广州 510515;2解放军总医院基础医学研究所,北京 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
Construction of fusion expression vector EGFP-PDX-1 and its transfection into rat fetal hepatic stem cells by electroporation
SUN Bing1; SUN Xiao-yan2; AN Jing1
1Department of Histology and Embryology, Southern Medical University, Guangzhou 510515, China; 2Institute of Basic Medical Sciences, General Hospital of PLA, Beijing 100853, China
Abstract: Objective To construct the fusion expression vector of pancreatic-duodenal homeobox gene 1 (PDX-1) fused to green fluorescent protein (GFP) capable of stable expression in fetal rat hepatic stem cells  after tranfection by electroporation. Methods PDX-1 cDNA was amplified from SK900/BLSCRIPT plasmid and cloned into the multiple cloning site of pEGFP-C1 to obtain the bined plasmid pEGFP-C1-PDX-1. Rat fetal hepatic stem cells were isolated, cultured, identified and transfected with the binant vector by electroporation, followed by observation of these cells with fluorescent microscope. The result of transfection was analyzed by RT-PCR and cell growth curve. Results Identification by enzyme digestion confirmed essful construction of the binant vector. Fetal hepatic stem cells can stably express GFP and PDX-1 for a period of time, and their growth and proliferation was not obviously affected after transfection. Conclusion The fusion expression vector of EGFP-PDX-1 is essfully constructed and stably expressed in rat fetal hepatic stem cells, which may facilitate the study of the role of PDX
-1 in stem cell di