血管内皮生长因子基因转染自体骨髓血管内皮祖细胞治疗下肢缺血的实验研究

An experimental study of autologous endothelial progenitor cells transfected with vascular endothelial growth factor(VEGF165) gene in the treatment of lower limb ischemia

目的:利用血管内皮生长因子(vascular endothelial growth factor,VEGF165)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高脂血日本大耳兔体内,观测其促进血管新生、改善肢体缺血的效果。方法:①制作高脂血兔,梯度离心法分离兔骨髓单个核细胞(MNCs),用内皮细胞专业培养基(EGM-2)诱导培养EPCs,并用双荧光染色法及免疫组化等方法进行鉴定。②脂质体介导携带EGFP标记的VEGF165质粒转染EPCs,用激光共聚焦显微镜检测VEGF蛋白的表达,用流式细胞仪检测转染率。③制作兔单侧下肢缺血模型,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及EGM-2培养基,多种方法检测移植效果。结果:①诱导出的梭形细胞,经FITC-UEA-I和DiI-acLDL荧光双染证实为正在分化的EPCs,同时经免疫组化法证实其Flk-1和Ⅷ因子的表达。②激光共聚焦显微镜下见绿色荧光蛋白表达,证实VEGF165转染成功,经流式细胞仪检测得到转染率约22.5%。③DSA及免疫组化检查显示VEGF165基因转染后的EPCs移植后改善肢体缺血的效果优于其他2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 explore the efficacy of the transplantation of autologous EPCs transfeeted with humanVEGF16s mediated by liposome in the treatment of lower limb ischemia. Methods： Make hypereholesterolemie rabbit model. Rabbit bone mar row mononuclear cells were isolated by dentity gradient eentrifugation. Mononuclear cells were cultivated with EGM-2 culture medium in order to induced them to EPCs, then identified with double fluorescence staining and immunohistochemistry. EGFP- N1-VEGF16transfeeted into EPCs mediated by lipofectamineTM2000. The expression of human VEGF15 gene was evaluated by laser scanning eonfocal microscope, VEGF transfection efficiency was detected by flow cytometry. We made unilateral hindlimb ischemia rabbit models . The rabbits were randomly divided into three groups which transplanted with EPCs, EPCs transfeeted with VEGF165（EPC/VEGF） and EGM-2 respectively, the efficacy of transplantation was evaluated by different ways. Results： （1）onuctear cells isolated were induced successfully. The double FITC-UEA- I and DiI-ac LDL fluorescence staining displayed differentiated EPCs, and immunohistoehemistry confirmed vascular endothelial growth factor receptor 2 and factor expression. （2）ong green fluorescence was observed in part of the cells after transfected with EGFP-N1-VEGF165plasmid 24 h,which indicated the success of transfeetion. The transduction efficiency in VEGF transduced EPCs was 22.5%detected by flow eytometry . （3）results of DSA and immunhistochemistry indicated that the effect of transplantation with EPCs transfected with VEGF16s were better than the other groups. There was significant difference between groups（ P 〈.01）. Conclusion：VEGF gene transfeetion can improve EPCs quality, the effect of transplantation for limb ischemic rabbits with EPCs transfected with VEGF are better than that with EPCs alone.