血管内皮生长因子165基因重组腺病毒载体的构建及转染内皮祖细胞的实验研究

目的：利用细菌内同源重组法构建含血管内皮生长因子（VEGF）165基因的重组腺病毒，并观察其在内皮祖细胞（EPCs）中的表达，为进一步研究其在慢性肾脏病中的作用奠定基础。方法：用限制性内切酶XbaⅠ＋HindⅢ从质粒载体中切出VEGFl65基因片段，与KpnⅠ＋HindⅢ双酶切的pAdTrack-CMV形成转移质粒pAdTrack-CMV-VEGFl65，PmeI酶切线性化后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183，由人胚肾293细胞包装成Ad-VEGFl65，PCR和westem blot法鉴定其表达。贴壁法体外培养获得大鼠骨髓来源的EPCs，Ad-VEGFl65基因体外转染EPCs，检测转染后目的基因的表达情况。结果：利用CSCI，法由pAdTrack-VEGFl65和pAdEasy-1共转化BJ5183感受态茵，可获得阳性重组体细菌克隆。PCR检测表明重组腺病毒已含有目的基因，病毒滴度1．4×10^10pfu／ml。Ad-VEGFl65转染培养第6天的EPCs后24h开始，培养上清中VEGF蛋白表达较对照组和转染前增加。结论：成功制备的重组体腺病毒Ad-VEGFl65转染体外培养的EPCs后，在体外能有效高表达目的基因产物，为今后VEGF在肾脏病中的深入研究奠定了基础。

Construction of Vascular Endothelial Growth Factor 165 Gene Recombinant Adenovirus and Transfection of Endothelial Progenitor Cells

Objective：To construct the recombinant adenovirus of vascular endothelial growth factor 165 （Ad-VEGF165） by homologous recombination in bacteria and to observe VEGF protein expression in rat endothelial progenitor cells derived from rat hone marrow transfected with Ad - VEGF165, Methods：VEGF165 gene was liberated from the vector via Xha Ⅰ and HindⅢ digestion,and subcloned into shuttle vector of pAdTrack- CMV plasmid digested by Kpn Ⅰ ＋ Hind Ⅲ, forming transfer vector of pAd-Track - CMV - VEGF165. Then pAdTrack - CMV - VEGF165 was linearized with Pine I and cotransformed into BJ5183 cells with adenovirus genomic plasmid of pAAEasy - 1. The DNA of identified recombinant plasmid was digested with Pac 1 and transfect- ed to 293 cells to package recombinant adenovirus particles. The polymerase chain reaction （PCR） was used to detect target gene. The titre of Ad- VEGF165 was measured with the aid of enhanced green fluorescent protein （EGFP） expression, EPCs derived from rat hone marrow were obtained by adherent culture for 6 days. After transfected by Ad - VEGF165, the expression of VEGF protein was assessed using Western blot. Results： Positive recombinant bacterial clones were obtained after co transformation of BJ5183 bacterial cells with pAdTrack - CMV - VEGF165 and pAdEasy - 1 by CsC12. PCR test indicated that the recombinant adenovirus contained the inserted VEGF165. The titre of purified recombinant adenovirus was 1,4 × 10^l0 pfu/ml, After transfection, the expression of VEGF protein was significantly higher than that of before transfected EPCs group and that of transfected with Ad - EGFP, Conclusion ： The conducted Ad- VEGF165 can effectively mediate target gene expression in cultured EPCs and this may paves a sound foundation for further study in chronic diseases.