摘要目的 探讨腺相关病毒(rAAV)介导的人血管内皮生长因子(VEGF)165基因转染大鼠骨髓间充质干细胞(MSC)移植对血管新生的影响.比较单纯干细胞移植与联合基因治疗的疗效.方法 全骨髓培养法提取培养MSC;rAAV-VEGF165转染MSC中,酶联免疫吸附试验(ELISA)及反转录-聚合酶链反应(RT-PCR)检测VEGF的表达;以近交系大鼠建立骨骼肌缺血模型,40只大鼠随机分为4组,每组10只.对照组注射磷酸盐缓冲液(PBS);干细胞组移植等量MSC,转染术后7 d组和转染术后10 d组分别于结扎7 d和10 d后的缺血区移植转染VEGF基因的MSC,移植6周后做Ⅷ因子染色检测血管新生情况.结果成功培养出MSC,免疫组化CD44阳性表达,CD34阴性表达;流式细胞术CD90阳性表达.在转染rAAV-VEGF165后转染组1、3、5、7、9 d上清液中VEGF165分泌水平分别为(131.98±6.00)、(263.96±4.58)、(540.85±5.97)、(208.98±5.06)、(174.45±5.00)ng/L,明显高于未转染组的(68.72±1.99)、(76.47±4.98)、(89.86±1.99)、(84.93±8.97)、(68.71±5.98)ng/L[t值分别为14.14、51.16、79.28、27.56、26.07,(均P＜0.05)],且5 d时达到高峰,此后表达开始下降.琼脂糖凝胶电泳可见在579 bp处见到高亮度条带,证明rAAV-VEGF165成功转染进MSC细胞中.转染后的生长曲线及细胞形态较未转染组无明显变化.Ⅷ因子染色示转染术后10 d组动物缺血区毛细血管密度[(9.35±2.72)条/视野]明显高于对照组[(1.05±0.50)条/视野]和干细胞组[(3.10±1.43)条/视野](均P＜0.01),较转染术后7 d组[(6.95±1.69)条/视野]亦有一定程度的升高(P＜0.05).结论 MSC有利于VEGF基因的稳定表达,可作为VEGF基因的良好细胞载体,且联合应用效果高于单独移植MSC,移植最佳时间为术后10 d.

abstractsObjective To evaluate the angiogenic effect of the bone marrow mesenchymal stem cells (MSCs) transfected with adeno-associated virus (rAAV) -mediated human vascular endothelial growth factor 165 (VEGF165) gene, to detect the expression and bioactivity of VEGF in the MSCs.and to detect the expression and bioactivity of VEGF165 gene in the area of ischemic skeletal muscle in rats. Methods The MSCs were cultured by whole bone marrow culture method, then the MSCs were transfected with rAAV-VEGF165, the expression of VEGF was examined using ELISA and RT-PCR. The model of ischemic skeletal muscle was established by ligation in inbred rats. The rats were injected with PBS at the ischemic zone (group A), MSC (group B), hVEGF-transfected MSC (group D), and 7 days later they were injected with hVEGF transfected MSC (group C). Six weeks after the transfection, the capillary density of the ischemic zone was examined by factor Ⅷ stain. Results The MSCs were cultured successfully according to the expressions of positive CD44 and CD90,negative CD34. In rAVV-VEGF165 transfected group, the secretion levels of VEGF165 in supernatant were significantly higher than in non-transfected group 1,3,5,7 and 9 days after injection [(131. 98±6.00) ng/L vs. (68. 72±1.99) ng/L; (263. 96±4.58) ng/L vs. (76. 47±4.98) ng/L;(540. 85±5.97) ng/L vs. (89. 86± 1.99) ng/L; (208. 98± 5.06) ng/L vs. (84. 93±8. 97) ng/L;(174.45±5.00) ng/L vs. (68.71±5.98) ng/L, all P＜0.05]. On the position of 579 bp, highbrightness strip could be seen. There were no significant differences in growth curve and cell morphology between transfected group and non-transfected group. Six weeks after the transplantation, the capillary density was significantly greater in group D [(9.35 ± 2.72)/ vision]than in group A [(1. 05±0. 50)/vision] ,group B [(3.10± 1.43)/vision, both P＜0. 01)] and group C [(6. 95± 1.69)/ vision, P＜0. 05] Conclusions MSCs are helpful for the stable expression of hVEGF gene, and it is good cellular vehicle for VEGF genes. The effect of combined therapy is much better than separate transplantation of MSCs. The best time to transplant is 10 days after surgical operation.