VEGF165和SDF-1双基因共表达腺病毒载体的构建及其在大鼠缺血脑组织中的表达

Construction of recombinant adenovirus vector co-expressing VEGF165 and SDF-1 genes and itsexpression in ischemic cerebral tissue of rats

目的构建血管内皮生长因子（VEGF）165与基质细胞衍生因子（SDF）-1双基因共表达腺病毒载体Ad5-VEGF165-IRES．SDF-1，并观察其在缺血大鼠脑组织中的共表达情况。方法将VEGF165和SDF-1基因通过内部核糖体进入位点（IRES）进行定向连接，以同源重组的形式构建双基因共表达重组穿梭质粒pDC316-VEGF165-IRES-SDF-1，将其与骨架质粒pBHGlox-E1以脂质体转染HEK293细胞，获得有感染能力的重组腺病毒颗粒，经多轮扩增后获得纯化的腺病毒载体Ad5-VEGF165．IRES．SDF．1；以线栓法构建大鼠大脑中动脉栓塞（MCAO）模型，并以立体定向微量注射的方法将构建的病毒载体注入大鼠侧脑室，以RT-PCR和免疫印迹法观察其介导VEGF165和SDF-1基因在缺血脑组织中的共表达情况。结果PCR、双酶切和基因测序等结果显示，重组质粒和腺病毒载体构建正确，并能够介导VEGF165和SDF-1两种基因在缺血的大鼠脑组织内共表达。结论成功构建了携带VEGF165和SDF-1双基因的腺病毒载体Ad5一VEGFl65一IRES-SDF-1，Ad5-VEGF165-IRES-SDF-1可以介导VEGF165和SDF-1双基因在缺血大鼠脑组织内共表达。

Objective To construct a recombinant adenoviral vector carrying and co-expressing vascular endothelial growth factor 165 （ VEGF165 ） and stromal cell derived factor 1 （ SDF-I ） and explore its co-expression in ischemic brain tissue in rats. Methods The VEGF165 and SDF-1 genes were directionally connected with internal ribosome entry site （IRES）. And the double gene coexpression recombinant shuttle plasmid pDC316-VEGF165-IRES-SDF-1 was built with homologous recombination. The resultant plasmid pDC316-VEGF165-IRES-SDF-1 and backbone plasmid pBHGlox El, 3Cre were transfeeted into HEK293 cells by liposome and the recombinant adenoviral particles capable of infection were aequirecL With the rounds of amplification, the purified adenoviral vector AdS-VEGF165-IRES-SDF-1 was obtained with a titer of up to 1 x 10^10 IU/ml. The rat model of middle cerebral artery occlusion （MCAO） was established by intra-luminal suturing. And the viral vectors were transfused into the lateral ventricle by a stereotactic microinjection. The expressions of VEGF165 and SDF-1 in iscbemic brain tissue were examined by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. Results The results of PCR, double enzyme digestion and gene sequencing showed that both the recombinant plasmid and the constructed adenoviral vector were expressed. And the adenoviral vector AdS-VEGF165-IRES-SDF-1 could mediate a co expression of VEGF165 and SDF-1 in isehemie cerebral tissue. Conclusion The recombinant adenoviral vector carrying VEGF165 and SDF-1 are successfully constructed. And AdS-VEGF165-IRES-SDF-1 maymediate a co-expression of VEGFI65 and SDF-1 in ischemic cerebral tissue of rats.