VEGF165基因转染EPCs移植对大鼠阿霉素肾病组织学的影响

Amelioration of Adriamycin nephropathy by transplanting endothelial progenitor cells transfected with Ad-VEGF165 gene

目的构建含血管内皮生长因子165基因的重组腺病毒(Ad-VEGF165),转染血管内皮祖细胞(EPCs)后,观察其对阿霉素肾病大鼠肾脏组织学的影响。方法用细菌内同源重组法构建含VEGF165基因的重组腺病毒Ad-VEGF165,通过PCR和Western blot鉴定所构建的腺病毒。贴壁法体外培养获得大鼠骨髓来源的EPCs,Ad-VEGF165体外转染EPCs,检测转染后目的基因的蛋白表达。大鼠阿霉素肾病模型形成过程中,尾静脉注射转染Ad-VEGF165的EPCs,在第16周时观察肾脏组织学变化。结果PCR和Western blot证实构建的重组腺病毒含有目的基因,滴度为1.4×1010PFU/ml;培养第6天的EPCs用于Ad-VEGF165转染,转染后24h,培养上清中VEGF蛋白表达较对照组和转染前增加。转染了VEGF165基因的EPCs移植后第12周(切肾术后16周),转染组的肾小球硬化和肾小管间质纤维化的程度明显较肾病组轻。结论转染了VEGF165基因的EPCs可以减轻阿霉素肾病的肾小球和肾小管的损伤程度,为VEGF在慢性肾脏病治疗提供了实验基础。

Objective To construct the recombinant adenovirus of vascular endothelial growth factor 165 （ Ad-VEGF165 ） and observe the effects of endothelial progenitor cells （EPCs） transfected with Ad-VEGF165 gene on ameliorating renal damages induced with Adriamycin. Methods Ad-VEGF165was identified with polymerase chain reaction （PCR） and Western blot. EPCs derived from rat bone marrow were obtained by adherent culture for 6 days and transfected with Ad-VEGF165, then injected into tail vein at the early stage of Adriamycin induced renal disease. Results PCR and Western blot indicated that the recombinant adenovirus contained the insert- ed VEGF165. The titer of the purified Ad-VEGF165 was 1.4×10^10 pfu/ml. After transfection, the expression of VEGF protein was significantly higher than that of control and untransfected EPCs. The pathological changes were less in VEGF165 transfecting group than that in untransfecting group at the 12^th week, including glomerular sclerosis and tubulointerstitial fibrosis induced by Adriamycin after transplantation. Conclusion The Ad- VEGF165 effectively mediates the target gene expression in cultured EPCs and ameliorates renal damages. But the adverse effect of VEGF to renal function needs further study.