Combination therapy of adeno-associated virus-mediated expression of vascular endothelial growth factor gene and bone marrow mesenchymal stem cells for limb ischemia
10.3760/cma.j.issn.0254-9026.2010.10.022
- VernacularTitle:腺相关病毒介导的血管内皮生长因子基因与骨髓间充质干细胞联合治疗大鼠肢体缺血的研究
- Author:
Fangjing WEI
;
Cuiyan WANG
;
Shuying YIN
;
Min XU
- Publication Type:Journal Article
- Keywords:
Vascular endothelial growth factors;
Myeloid progenitor cells;
Dependovirus
- From:
Chinese Journal of Geriatrics
2010;29(10):858-862
- CountryChina
- Language:Chinese
-
Abstract:
Objective 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.
