HGF基因转染促进同种瓣移植后体内再内皮化研究

Promotion of in vivo reendothelialization by HGF gene transfection after homograft transplantation

目的探讨同种瓣经去内皮和肝细胞生长因子(HGF)转染处理后,不同移植时间点的再内皮化效果及CD137、Gab1表达情况。方法成年健康近交系中国雄性家兔为供体,近交系成年新西兰雌性大白兔为受体,行同种瓣补片法移植。受体随机分为3组,每组20只,新鲜组不做任何处理,作为对照;去内皮组行去内皮细胞处理;HGF组医用生物蛋白胶携载HGF质粒,对去内皮处理的移植物进行基因转染。分别于术后2、4、8、12周取出移植物,移植物行光镜与电镜观察组织形态变化,免疫组化法测定CD137与移植后2周Gab1的表达。结果去内皮组与新鲜组分别于移植后4周与8周出现新生内皮细胞,早期伴有不同程度的平滑肌细胞损伤;术后2周,HGF组移植物表面即出现大量内皮细胞覆盖,且无平滑肌细胞钙化坏死。新鲜组与去内皮组4、8、12周3个不同时间点的CD137表达较HGF组显著升高,差异有统计学意义(P<0.01);HGF组样本中,移植物各时间点CD137表达情况比较,差异无统计学意义(P>0.05)。3组CD137的表达高峰均出现在第2周。移植2周时,HGF组Gab1表达量(0.198 2±0.024 3)明显高于新鲜组(0.107 7±0.010 4)与去内皮组(0.110 2±0.014 5),差异有统计学意义(P<0.01);新鲜组与去内皮组Gab1表达量比较,差异无统计学意义(P>0.05)。结论去内皮及HGF基因转染处理同种瓣能够上调Gab1的表达,在移植早期(2周)实现体内再内皮化,同时抑制早期免疫反应。

Objective To explore the changes in tissue morphology,CD137 and Gab1 expression in heterotropic transplantation after treatment of aortic valved homograft with endothelium removal and transfection of hepatocyte growth factor （HGF）. Methods The Healthy,inbred adult Chinese male rabbits were used as donors, and inbred adult New Zealand white female rabbits as recipients. Abdominal aorta heterotopic transplantation with aortic valved homograft was performed. Recipients were randomly divided into 3 groups, with 20 in each group, no treatment was appliedfor the aortic valved homograft in the control group ; endothelial cell removal was applied for the aortic valved homograft in the endothelium removal group, and gene transfection was performed on the graft after endothelium removal with HGF plasmid carried by fibrin glue in the HGF group. The grafts were obtained 2,4,8 and 12 weeks after the surgery. Changes in tissue morphology of the graft were observed by light and electron microscopy was measured by flame atomic absorption spectrometry, Gabl （2 weeks after the surgery）and CD137 expression were measured by immunohistochemical assay. Results Following various degrees of smooth muscle cells injury after transplantation,new endothelial cells appeared at 8 weeks in fresh group and at 4 weeks in endothelium removal group;2 weeks after operation, the grafts in HGF group were mostly covered with new endothelial cells, no calcification or necrosis of smooth muscle cells was observed. The CD137 expression at four time points--week 2, week 4, week 8 and week 12 in the fresh and endothelium removal groups were significantly increased compared with the HGF group（P 〈0. 01 ） ;the change in CD137 expression showed no significant difference at all time points in the graft samples of the HGF group（ P〉0. 05 ）. The peak of CD137 expression appeared in the second week in the three groups. At week 2,the Gabl expression in the HGF group（0. 198 2±0. 024 3） was significantly higher than that in the fresh（0. 107 7 ＋0. 010 4）and endothelium removal groups（0. 110 2 ＋0. 014 5）, （P 〈0. 01 ） ; there was no statistically significant difference of Gabl express quantity between the fresh group and the endothelium removal group, （ P 〉 0.05 ）. Conclusion In vivo reendothelialization can be achieved in the early stage of transplantation （ week 2 ） via endothelium removal and HGF gene transfection,which can up-regulating the expression of Gabl to inhibite early immune response.