血管内皮生长因子基因转染骨髓间充质干细胞对慢性肾衰竭大鼠血管新生的影响

目的：探讨血管内皮生长因子（ vascular endothelial growth factor ， VEGF）基因转染骨髓间充质干细胞（ mesenchy-mal stem cells，MSCs）对慢性肾衰竭（chronic renal failure， CRF）大鼠肾小球内皮细胞修复和血管新生的影响。方法体外分离、培养大鼠MSCs，Ad5-hVEGF-EGFP转染MSCs并观察转染对细胞增殖，分泌VEGF的影响。50只雄性SD大鼠按数字表法随机分为假手术组、慢性肾衰竭组、MSCs移植组、Ad-VEGF注射组和VEGF基因转染的MSCs移植组，每组10只。采用分阶段5／6肾切除术制备大鼠慢性肾衰竭动物模型。假手术组与慢性肾衰竭组于切除右肾之前从该侧肾动脉注射不含血清的改良杜氏伊格尔（DMEM）培养液，其余3组分别给予MSCs，Ad-VEGF和VEGF基因转染的MSCs。8周后检测各组大鼠肾功能，并用免疫组化检测肾小球CD31表达情况。结果 Ad5-hVEGF-EGFP转染后对MSCs细胞增殖无影响，转染后分泌VEGF蛋白较空质粒转染组明显增加（P＜0.05）。 MSCs移植组和VEGF基因转染的MSCs移植组血肌酐（Scr）和尿素氮（BUN）均较慢性肾衰竭组降低（P＜0．05）；与MSCs移植组比较，VEGF基因转染的MSCs移植组Scr和BUN降低更显著（P＜0．05）。慢性肾衰竭组肾小球中CD31表达较假手术组显著降低（P＜0．05），应用MSCs和VEGF165基因转染的MSCs干预后肾小球中CD31表达有所增加，VEGF基因转染的MSCs组增加更明显（P＜0．05）。结论慢性肾衰竭大鼠给予转染VEGF基因的MSCs移植后毛细血管内皮细胞数量增多，肾脏功能改善；其作用可能与基因转染增加VEGF表达及有利于肾小球毛细血管内皮修复、血管新生有关。

Effects of bone marrow mesenchymal stem cell gene transfection by VEGF gene on angiogenesis in rats with chronic renal failure

Objective To investigate the effects of bone marrow mesenchymal stem cell （MSCs） gene transfection by VEGF （vascular endothelial growth factor ） gene on the repair of glomerular endothelia and angiogenesis in rats with chronic renal failure （CRF）. Methods MSCs isolated from rat bone marrow were cultured and transfected with recombinant Ad5-hVEGF-EGFP.Then, effects of transfection on cell proliferation and secretion of VEGF were observed closely .Fifty male SD rats were randomly divided into 5 groups：the sham operation group , the CRF group, the MSCs transplantation group , the Ad-VEGF injection group and the MSCs transfected by VEGF gene group.The animal CRF model was established by a two-stage 5/6 nephrectomy procedure in rats .For the animals of the sham opera-tion group and the CRF group , DMEM culture solution without serum was injected through the right renal artery prior to nephrectomy of the right kidney.The animals of the other 3 groups were treated with MSCs , Ad-VEGF and MSCs transfected by VEGF genes.After 8 weeks, the renal function of all the groups was detected and CD 31 expression in glomerulus was measured by immunohistochemistry .Results The effect of Ad5-hVEGF-EGFP on the proliferation of MSCs could not be detected after transfection .The level of VEGF protein in supernatant after VEGF gene transfection was significantly higher than that of the control group （P〈0.05）.The Scr and BUN levels in the MSCs transplantation group and the MSCs transfected by VEGF gene group were lower than those of the CRF group （P〈0.05）.As compared with those of the MSCs transplantation group , the levels of Scr and BUN for the MSCs transfected by VEGF gene group were even lower （P〈0.05）.CD31 expression in glomerulus for the CRF group was decreased , as compared with that of the sham operation group （P〈0.05）.However, CD31 expression in glomerulus was increased to some extent following treatment by MSCs or MSCs transfected by VEGF gene （P〈0.05）.CD31 expression level was increa