aFGF重组腺相关病毒转染内皮祖细胞  

Recombinant adeno-associated virus encoding secreted forms of human acidic fibroblast growth factor transfecting endothelial progenitor cells

作　　者：陈书艳[1] 颜雪芸[1] 王飞[1] 周卿[1] 荣烨之[1] 

机构地区：[1]上海交通大学医学院新华医院心血管内科,上海200092

出　　处：《基础医学与临床》2006年第10期1067-1071,共5页Basic and Clinical Medicine

基　　金：国家自然科学基金(30270543)

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20％-30％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560bp的基因条带，通过Western blot检测到被感染细胞中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。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 infected EPC was observed by fluorescence microscope and expression of sp-haFGF in EPCs was verified by RT-PCR and western blot analysis. Results Recombinant AAV encoding sp-haFGF was obtained. After EPCs being infected with rAAV, green fluorescence was found in about 20 - 30% EPCs, gene of 560 bp was generated from EPCs by RT-PCR method, and sp-haFGF protein was detected in infected cells. Conclusion EPC was efficiently infected by rAAV encoding sp-haFGF and haFGF was expressed by EPCs, which establishes a basis for therapeutic angingenesis by transgenic EPCs transplantation.

关 键 词：酸性成纤维细胞生长因子 腺相关病毒 内皮祖细胞 

分 类 号：Q78[生物学—分子生物学]