腺相关病毒介导的人血管内皮生长因子体外转染兔骨髓内皮祖细胞

Adenovirus-associated Virus Mediated Human Vascular Growth Factor Gene Transfer in Rabbit Bone Marrow Derived Endothelial Progenitor Cells in Vitro

目的:探讨腺相关病毒-2(AAV-2)介导人血管内皮生长因子-165(hVEGF165)转染兔骨髓内皮祖细胞(EPCs)及其对EPCs增殖能力的影响。方法:构建携带hVEGF165基因的重组腺相关病毒(rAAV)载体pAAV-hVEGF165;制备携带hVEGF165基因的rAAV(rAAV-2-hVEGF165)和携带β-半乳糖苷酶(-βgal)基因的rAAV(rAAV-2-LacZ);体外培养和鉴定兔骨髓EPCs;分别用rAAV-2-hVEGF165和rAAV-2-LacZ体外转染EPCs(AAV/VEGF-EPC和AAV/LacZ-EPC);用RT-PCR、免疫细胞化学、流式细胞术等法检测hVEGF165和-βgal的表达,MTT法检测AAV/VEGF-EPC的增殖能力。结果:经酶切鉴定和DNA测序,得到了序列正确的重组质粒pAAV-hVEGF165;AAV/VEGF-EPC能够表达hVEGF165蛋白,AAV/LacZ-EPC能够表达-βgal;rAAV-2-hVEGF165对于EPCs的转染率为64.4%;AAV/VEGF-EPC的增殖能力明显强于AAV/LacZ-EPC和EPCs。结论:EPCs可以被rAAV-2-hVEGF165高效转染,其增殖能力明显增强。本文为进一步探讨AAV/VEGF-EPC用于缺血性血管疾病治疗的可能性奠定了基础。

Objective： To study adenovirus-associated virus-2 （AAV-2） mediated human vascular growth factor 165 （hVEGF165） transfecting rabbit bone marrow derived endothelial progenitor cells （EPCs） in vitro, and the effect on EPCs＇ proliferation. Methods： A recombinant adeno-associated virus （rAAV） vector encoding human VEGF165 gene （pAAV-hVEGF165） was constructed. The rAAV vectors encoding hVEGF165 （rAAV2-hVEGF165） or β-galactosidase （rAAV2-LacZ） were prepared. EPCs were cultured and identified in vitro. EPCs were trasfected by rAAV2-hVEGF165 （AAV/VEGF-EPC） or rAAV2-LacZ （AAV/LacZ-EPC） respectively. Expression of hVEGF165 and β-galactosidase （β-Gal） were detected by RT-PCR, immunocytochemistry and flow cytometry methods. The proliferation of AAV/VEGF-EPC was measured by MTT assay. Results： pAAV- hVEGF165 was verified by enzyme digestion and DNA sequencing, rAAV2-hVEGF165 could transfeet EPCs with an efficiency of 64.4%. AAV/VEGF-EPC could express hVEGF165 and AAV/ LacZ-EPC could express β-gal. The proliferation potential of AAV/VEGF-EPC was significantly higher than that of AAV/LacZ-EPC. Conclusion： rAAV2- hVEGF165 could efficiently transfect EPCs and enhance their proliferation potential. AAV/VEGF-EPC could be used for further research on therapy of ischemic disease.