血管内皮生长因子基因转染血管内皮祖细胞治疗肢体缺血的实验研究

An experimental study of endothelial progenitor cells transfected with VEGF165 gene in the treatment of limb ischemia

目的利用血管内皮生长因子（vascular endothelial growth factor,VEGF-165）基因转染体外诱导的血管内皮祖细胞（endothelial progenitor cells,EPCs）,并移植到下肢缺血的新西兰兔体内,观测其促进血管新生,改善肢体缺血的效果。方法（1）梯度离心法分离兔骨髓单个核细胞,然后用含有VEGF、bFGF、IGF-1的M199培养液诱导培养EPCs。并以免疫荧光、透射电镜等方法进行鉴定。（2）用携带VEGF165基因的腺病毒质粒（Adv-GFP-VEGF165）转染所培养的细胞,ELISA法检测上清液中VEGF蛋白的表达。（3）制作兔下肢缺血模型,并将其随机分为A、B、C 3组,分别移植EPCs、VEGF165基因转染后的EPCs、M199培养基,多种方法检测移植效果及局部整合情况。结果（1）自兔骨髓诱导出梭形贴壁细胞,免疫荧光及电镜检测证实为EPCs。（2）Adv-GFP-VEGF165成功转染EPCs,ELISA法检测转染VEGF165后的EPCs其上清液中VEGF蛋白浓度明显升高。（3）Brdu示踪显示移植细胞整合到缺血局部,CTA及免疫组化检查显示VEGF-165基因转染后的EPCs移植后其改善肢体缺血效果优于其他两组。结论VEGF基因转染EPCs后能改进EPCs质量,移植后促血管新生能力增强,其效果优于未转染组。

Objective To observe the effects of neovascularization in limb ischemic rabbits transplanted with EPCs transfected with VEGF165. Methods （ 1 ） Rabbit bone marrow mononuclear cells were isolated by gradient centrifugation,then cultivated with M199 culture medium and induced to EPCs by VEGF, bFGF and IGF-1. Cultivated cells were identified by immunofluorescence and electron microscope. （2） EPCs were transfected with Adv-GFP-VEGF165. The VEGF gene expression was observed by detecting the concentration of VEGF protein in supernatant with ELISA method. （3）Rabbit hind limb ischemia models were produced and rabbit model was divided into three groups which transplanted with EPCs, EPCs transfected with VEGF165 and M199 respectively. The effects of transplantation and incorporation were observed. Results （1） EPCs were induced successfully, which was confirmed by immunofluorescence and electron microscope. （2） EPCs were transfected with VEGF165 successfully. The VEGF protein concentration in the supernatant of transfected group was higher than that of control group observed with ELISA method. （3）Transplanted cells incorporated into ischemic region which was confirmed by Brdu. The results of CTA and immunohistochemistry indicated that the effects of transplantation with EPCs transfected with VEGF165 were better than other groups. Conclusion VEGF gene transfection can improve EPCs quality, the effects of transplantation for limb ischemic rabbits with EPCs transfected with VEGF were better than with EPCs alone.