重组人酸性成纤维细胞生长因子的构建及转染

基础医学与临床 ›› 2006, Vol. 26 ›› Issue (10): 1067-1067.

重组人酸性成纤维细胞生长因子的构建及转染

陈书艳颜雪芸王飞

上海第二医科大学新华医院上海上海第二医科大学新华医院上海上海第二医科大学新华医院上海

收稿日期:2005-07-22 修回日期:2006-04-04 出版日期:2006-10-25 发布日期:2006-10-25
通讯作者: 陈书艳

Construction of a recombinant human acidic fibroblast growth factor and its expression

Received:2005-07-22 Revised:2006-04-04 Online:2006-10-25 Published:2006-10-25

摘要/Abstract

摘要： 目的研究含分泌型人酸性成纤维细胞生长因子(sp-haFGF)基因的重组腺相关病毒（rAAV）感染内皮祖细胞(EPCs)的可行性。方法用PCR法将FGF-4的信号序列与原始的aFGF基因连接，形成sp-haFGF基因。将此目的基因克隆到AAV载体质粒pAAV-IRES-hrGFP中，并与包装质粒(pAAV-RC)和辅助质粒(pHelper)共转染HEK293细胞，获得编码sp-haFGF的重组腺相关病毒。用浓缩的病毒感染体外培养的EPC，荧光显微镜下观察细胞表达绿色荧光蛋白情况,并用RT-PCR和western blot方法鉴定sp-haFGF在EPC中的表达。结果获得含有sp-haFGF的重组腺相关病毒，将其感染EPC后，约30～40%的细胞出现绿色荧光。用RT-PCR法从被感染细胞中扩增出一条长约550bp的基因条带，通过western blot检测到被感染细胞中sp-haFGF蛋白的表达。结论编码sp-haFGF的重组腺相关病毒能够有效地感染EPC，并在EPC中表达sp-haFGF，为进一步研究转基因的内皮祖细胞移植促血管新生奠定基础。

Abstract: Objective To investigate the feasibility of recombinant adeno-associated virus encoding secrected forms of human acidic fibroblast growth factor transfecting endothelial progenitor cells. Methods sp-haFGF was obtained through combining signal peptide sequence of FGF-4 with native aFGF gene by PCR. Sp-haFGF was cloned into AAV vector plasmid pAAV-IRES-hrGFP . Recombinant AAV encoding sp-haFGF was packaged through co-transfecting HEK293 cells with plasmid sp-haFGF-pAAV-IRES-hrGFP, pAAV-RC and pHelper. Ex vivo cultured EPCs were infected with concentrated rAAV . Expression of green fluorescent protein(GFP) in EPC infected was observed by fluorescence microscope and expression of sp-haFGF in EPCs was verified by RT-PCR and western blot analysis. Results Recombinant

陈书艳颜雪芸王飞. 重组人酸性成纤维细胞生长因子的构建及转染[J]. 基础医学与临床, 2006, 26(10): 1067-1067.

. Construction of a recombinant human acidic fibroblast growth factor and its expression[J]. Basic & Clinical Medicine, 2006, 26(10): 1067-1067.