Wang Sheng, Chen Zhong, Tang Xiaobin, Liu Hui, Yang Liao, Wang Yanyang
Department of Vascular Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, P.R. China.
Mol Med Rep. 2015 Oct;12(4):4967-74. doi: 10.3892/mmr.2015.4100. Epub 2015 Jul 20.
The present study aimed to investigate the effects of neovascularization in rabbits with limb ischemia transplanted with vascular endothelial growth factor (VEGF)165‑transfected endothelial progenitor cells (EPC). Bone marrow mononuclear cells were isolated by gradient centrifugation, cultured in M199 culture medium and induced into EPCs using VEGF, basic fibroblast growth factor, and insulin‑like growth factor‑1, and subsequently identified. The EPCs were transfected with Adv‑green fluorescent protein‑VEGF165 and the proliferation potential of the cells was determined using an MTT assay. The protein expression levels of VEGF were measured by detecting its concentration levels in the supernatant using an ABC‑ELISA assay. A rabbit hind limb ischemic model was established and randomly divided into three groups: (A) Control group, (B) EPC‑transplanted group, and (C) Ad‑VEGF165/EPCs‑transplanted group. The effects of transplantation and the levels of recanalization were detected. Incorporation of the transplanted cells into the ischemic region was confirmed by 5‑bromodeoxyuridine staining, and the levels of recanalization were measured by computer tomography ateriography and immunohistochemical staining. Bone marrow‑derived EPCs were induced, cultivated, and successfully identified. The results of the present study determined the optimum transfection ratio that promoted the growth of EPCs. The EPCs were successfully transfected with VEGF165, and EPC proliferation was not affected by the transfection. The supernatant protein concentration levels of VEGF were markedly higher in the VEGF165‑transfected group, as compared with those of the control group. Introduction of the transplanted cells into the ischemic region of group C occurred more efficiently, as compared with groups A and B. The recanalization capillary density in group C was significantly higher, as compared with groups A and B. VEGF gene transfection was able to improve the quality of EPCs, and the response of rabbits with limb ischemia to transplantation with VEGF‑transfected EPCs was significantly better, as compared with transplantation with EPCs alone.
本研究旨在探讨血管内皮生长因子（VEGF）165转染的内皮祖细胞（EPC）移植对兔肢体缺血新生血管形成的影响。采用梯度离心法分离兔骨髓单个核细胞，接种于M199培养基中，并用VEGF、碱性成纤维细胞生长因子和胰岛素样生长因子-1诱导分化为EPC，随后进行鉴定。将携带绿色荧光蛋白的腺病毒载体VEGF165转染EPC，采用MTT法检测细胞增殖能力。采用ABC-ELISA法检测上清液中VEGF浓度，以测定其蛋白表达水平。建立兔后肢缺血模型，随机分为3组：（A）对照组、（B）EPC移植组和（C）Ad-VEGF165/EPCs移植组。检测移植效果及再通水平。用5-溴脱氧尿苷染色证实移植细胞整合到缺血区域，并用计算机断层血管造影和免疫组织化学染色测量再通水平。成功诱导、培养并鉴定出兔骨髓来源的EPC。本研究结果确定了促进EPC生长的最佳转染率。VEGF165成功转染EPC，且转染不影响EPC增殖。与对照组相比，VEGF165转染组上清液中VEGF蛋白浓度明显升高。与A组和B组相比，C组移植细胞更有效地整合到缺血区域。与A组和B组相比，C组再通毛细血管密度明显更高。VEGF基因转染能够改善EPC质量，与单纯EPC移植相比，VEGF转染的EPC移植对兔肢体缺血的反应明显更好。
