中文摘要：

背景：目前用于基因治疗的病毒载体对机体具有一定免疫原性，且存在潜在感染危险，限制了临床的应用。 目的：拟构建带有绿色荧光蛋白标记并携带hVEGF165基因的无致病性重组腺相关病毒，以其作为基因载体感染兔骨髓基质干细胞，体外检测病毒生物学活性及功能。 设计、时间和地点：开放性实验，于2007—03／11在陕西省疾病预防控制中心病毒室完成。 材料：兔骨髓基质干细胞由试验者原代培养，取自成年新西兰大白兔：人脐静脉内皮细胞取自健康产妇婴儿脐带，产妇对本实验知情同意。 方法：从含有hVEGF165基因的pUC18-hVEGF165质粒中扩增出hVEGF165片段，构建重组骨架质粒pAAV—hVEGF165—IRES—hrGFP。将此质粒和腺相关病毒包装质粒pAAV—RC、辅助质粒pAAV—Helper共转染AAV-293细胞，通过同源重组产生重组腺相关病毒rAAV—hVEGF165-GFP。应用该重组病毒感染体外培养的兔骨髓基质干细胞。 主要观察指标：荧光显微镜下监测病毒包装效率，感染AAV—HT1080测定病毒滴度，并通过病毒基因组外源基因扩增鉴定重组病毒的包装是否成功。检测重组病毒的感染效率。ELISA法检测培养上清中血管内皮生长因子含量，人脐静脉内皮细胞管状血管形成试验检测血管内皮生长因子蛋白活性。 结果：扩增产物经DNA测序确定为hVEGF165 cDNA片段，重组骨架质粒pAAV—hVEGF165-IRES—hrGFP经双酶切鉴定正确。病毒包装效率达95％以上，收获纯化病毒滴度达5．5×10^11vg／mL。提取重组病毒基因组成功扩增出外源目的基因片段，证实重组病毒rAAV-VEGF165-GFP包装成功。重组病毒rAAV-VEGF165-GFP感染骨髓基质干细胞效率为40％，感染72h后骨髓基质干细胞中血管内皮生长因子含量达（85．58±537）μg／L，分泌的血管内皮生长因子蛋白可以使人脐静脉内皮细胞拉长变形、出芽且相互交联成管?

英文摘要：

BACKGROUND： Presently, viral vector used in gene therapy has immunogenicity to the body and has a potential to infect, so it has limitation to be used in clinic. OBJECTIVE： To construct the non-pathogenic recombinant adeno-associated virus （AAV） simultaneously carrying human vascular endothelial growth factor 165 （hVEGF165） gene and green fluorescent protein （GFP） label, and assess its biological activity and function by infecting rabbit bone marrow mesenchymal stem cells （BMSCs） in vitro. DESIGN, TIME AND SETTING： An open experiment was performed at the Laboratory of Virus of Shanxi Provincial Center for Disease Control and Prevention between March and November 2007. MATERIALS： BMSCs were harvested from mature New Zealand rabbits after primary culture by the experimenter. Human umbilical vein endothelial cells （HUVECs） were harvested from infant umbilical cores of healthy parturients. Informed consents were obtained from parturients. METHODS： Plasmid pAAV-hVEGF165-IRES-hrGFP was constructed by hVEGF165 segments amplified from plasmid pUC18-hVEGF165 carrying hVEGF165 gene. The recombinant expression plasmid pAAV-hVEGF165-IRES-hrGFP was co-transfected into AAV-293 cells with pAAV-Helper and pAAV-RC for recombinant AAV replication and package through homologous recombination. In vitro cultured rabbit BMSCs were infected with this recombinant virus. MAIN OUTCOME MEASURES： The efficiency of AAV packaging was monitored under a fluorescent microscope and the virus titer was measured through infecting AAV-HT1080, and the recombinant virus was verified by polymerase chain reaction （PCR） of the exogenous interest gene. Through infecting rabbit bone marrow mesenchymal stem cells in vitro, the infection efficiency was detected. The expression of VEGF165 in BMSCs was detected by enzyme linked immunosorbent assay （ELISA）, and its biological activity was assessed by angiopoiesis experiment of HUVECs in vitro. RESULTS： The hVEGF165gene was successfully amplified and recombin