课题背景：课题属于国家自然科学基金项目(30600624)部分研究内容，目前已经完成重组腺相关病毒载体的包装及鉴定，并完成体外感染兔骨髓基质干细胞功能检测的初步应用试验。

应用要点：①以腺相关病毒作为血管内皮生长因子基因载体，具有无致病性、免疫原性低及宿主范围广、能够感染多种宿主细胞并长期表达等多种优点，克服了目前基因治疗中所采用的质粒脂质体载体转染率低、腺病毒等病毒载体对机体具有一定免疫原性、存在潜在感染危险等缺点。②重组载体同时携带标记基因绿色荧光蛋白，可以方便在病毒包装、纯化过程中监测包装效率及病毒感染效率，并利于后期重组病毒感染宿主细胞后的定位跟踪观察。

偏倚或不足：获得大量重组病毒需反复多次包装、纯化，不利于同期大规模开展，而多次进行包装所获得的病毒粗提液内病毒的含量及滴度可能存在不同，需要在后期进行统一调整。实验获得的携带人血管内皮生长因子基因的高滴度重组腺相关病毒总量仍有不足。重组病毒体外感染骨髓基质干细胞后功能测定还需要从更多角度补充完善。

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5.5×1011 vg/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72 h后骨髓基质干细胞中血管内皮生长因子含量达（85.58±5.37）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状样结构。
结论：成功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rAAV-VEGF165-GFP，该重组病毒

能够高效感染兔骨髓基质干细胞并表达血管内皮生长因子，分泌的血管内皮生长因子蛋白具有良好的生物活性，可有效促进人脐静脉内皮细胞的增殖。
关键词：骨髓基质干细胞；血管内皮生长因子；腺相关病毒；绿色荧光蛋白

黄向辉，时志斌，王坤正，党晓谦，杨佩，余鹏博.重组hVEGF165腺相关病毒载体构建及体外感染骨髓基质干细胞血管内皮生长因子的表达[J].中国组织工程研究与临床康复，2008，12(21):4097-4101
[www.zglckf.com/zglckf/ejournal/upfiles/08-21/21k-4097(ps).pdf]

Construction of recombinant adeno-associated virus carrying human vascular endothelial growth factor 165 and in vitro infection of bone marrow mesenchymal stem cells

Abstract

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 hVEGF165 gene was successfully amplified and recombinant pAAV-hVEGF165-IRES-GFP was verified by double digestion. The system provided a high packing ratio of more than 95% and the purified recombinant virus had a high titer of 5.5×1011 vp/mL. The recombinant virus was confirmed by exogenous human VEGF165 gene PCR. The rAAV-VEGF165-GFP could infect rabbit BMSCs at a ratio of 40%. With rAAV-VEGF165-GFP infection, the expression of VEGF165 in the conditional medium was detected by ELISA at a concentration of（85.58±5.37）μg/L 72 hours after infection. HUVECs became extended, deformed, pullulation, cross-linking and showing tube-shape affected by VEGF protein.
CONCLUSION: The non-pathogenic rAAV-hVEGF165-GFP simultaneously carrying hVEGF165 and GFP label is successfully constructed, with the ability of infecting rabbit BMSCs to efficiently express hVEGF165，and hVEGF165 expressed in BMSCs has a satisfying biological activity of stimulation of HUVECs proliferation.

Huang XH, Shi ZB, Wang KZ, Dang XQ, Yang P, Yu PB.Construction of recombinant adeno-associated virus carrying human vascular endothelial growth factor 165 and in vitro infection of bone marrow mesenchymal stem cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(21):4097-4101(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-21/21k-4097(ps).pdf]