转染人VEGF165基因对大鼠胰岛移植物再血管化及生物学功能的影响

目的探讨转染人血管内皮生长因子（VEGF）基因对大鼠胰岛移植物再血管化和生物学功能的影响。方法实验分二步进行，首先构建携带人血管内皮生长因子165（hVEGF165）基因的重组腺病毒载体AdhVEGF165，用其按病毒／细胞的感染倍数（MOI）分别为10、100、500的比例在体外转染新鲜分离、纯化的近交系Lewis大鼠胰岛，检测体外培养的胰岛表达hVEGF165基因的情况；然后按MOI为10的比例在体外用AdhVEGF165转染Lweis大鼠胰岛，再经门静脉注射至近交系Lewis大鼠的肝脏内（VEGF组），并设不含hVEGF165基因的空载体转染对照组（GFP组）和磷酸盐缓冲液处理对照组（PBS液组），术后测定受鼠的血糖变化，进行静脉糖耐量试验（IVGTT），并观察受鼠肝脏中移植胰岛的组织学变化。结果体外转染hVEGF165基因的大鼠胰岛分泌至细胞外的hVEGF165的浓度显著高于未转染者（P〈0．01）。糖尿病大鼠移植转染hVEGF165基因的胰岛后，静脉注射葡萄糖后40、60、120min时的血糖水平显著低于GFP组和PBS液组（P〈0．05）；40min时体循环中胰岛素的浓度，VEGF组显著高于GFP组和PBS液组（P〈0．05）；VEGF组移植胰岛内CD34和胰岛素免疫组化染色的强度均高于GFP组和PBS液组。结论转染hVEGF165基因对胰岛移植物的再血管化和生物学功能有促进作用。

Effects of hVEGF165 gene transfection to pancreatic islets on the revascularization and function of engrafted inbred rats'' islets posttransplantation

Objective To investigate the effects of the hVEGF165 gene transfection on the revascularization and function of syngeneic pancreatic islets posttransplantation.Methods The recombinant replication-deficient adenovirus vector named AdhVEGF165 was constructed,which carried the hVEGF165 gene.Freshly isolated islets of SD rats were tansfected with AdhVEGF165 in vitro.The expression of hVEGF165 was detected with ELISA Kit in vitro.Finally the transfected islets of inbred Lewis rats were transplanted into the liver of syngeneic diabetic rats via portal vein.Nonfasting blood glucose(NBG) was determined posttransplantation.Intravenous glucose tolerance test(IVGTT) was performed 3 weeks later.Serum insulin was measured with radioimmunologic Kit during IVGTT.The livers of recipients were resected,then the pathological examination was carried out,including the immunohistochemical analysis.Results Islets transfected with AdhVEGF165 expressed hVEGF165,moreover the quantity of hVEGF165 in VEGF group was much higher than in the two control groups.IVGTT revealed that the blood glucose concentration was decreased faster in recipients with islets overexpressing hVEGF165 and the serum insulin concentration was significantly higher in the group VEGF.Islets overexpressing hVEGF165 showed higher vascular density pathologically.Conclusions With islets overexpressing hVEGF165,the revascularization was improved after syngeneic transplantation.Islets overexpressing hVEGF165 exhibited more powerful capability of sustaining the systemic homeostasis of glucose metabolism.