携带人血管内皮细胞生长因子基因细菌内重组腺病毒转染兔骨髓基质干细胞的表达

目的应用高效细菌内同源重组系统pAd-Easy,制备含人血管内皮细胞生长因子121 (VEGF121)基因的重组腺病毒,感染兔骨髓基质干细胞(BMSCs),并检测外源基因的表达,为其进一步应用于血管化组织工程骨组织的构建打下基础。方法自pCDNA-VEGF121质粒中切取VEGF121基因,将VEGF基因克隆到穿梭质粒,在BJ5183细菌内重组,在293细胞中构建携带VEGF基因的重组腺病毒,将腺病毒感染BMSCs,利用ELISA、免疫组织化学的方法检测VEGF121在BMSCs中的表达。结果通过pAd-Easy系统成功构建高滴度的携带VEGF121基因重组腺病毒pAd-VEGF,ELISA检测显示经转染的BMSCs中VEGF121的表达均明显增强,随着感染病毒MOI值的增高,VEGF121的表达量相应增高,差异有统计学意义(P<0．01),免疫组化法显示经基因转染的BMSCs中有强阳性信号,未转染组出现阴性结果。结论Ad-VEGF转染BMSCs后能稳定提高VEGF蛋白的表达。

Expression of vascular endothelial growth factor in bone marrow stem cells transferred by recombinant adenovirus homologously recombined in bacteria

Objective To transfect the recombinant adenovirus with human vascular endothelial growth facter(VEGF) gene into rabbit bone marrow stem cells and study the VEGF expression in bone marrow stem cell (BMSCs) . Methods We used a new bacterial homologous recombination system (pAd-Easy system) to construct recombinant adenovirus vectors. The VEGF gene was cut down from the PCDNA-VEGF plasmid and then transferred to track plasmid. Recombination was successfully completed in bacteria BJ5183 and the recombinant adenovirus was produced in 293 cells. Next we infected BMSCs and explored the expression of VEGF protein by means of ELISA and immunohistochemistry. Results We successfully constructed the recombinant adenovirus with VEGF gene and detected yellow and brown particles in BMSCs transferred by VEGF gene. Conclusion The recombinant adenovirus with VEGF gene can transfect the VEGF gene into BMSCs and highly enhance the expression of VEGF protein.