正、反义VEGF基因转染对人增生性瘢痕成纤维细胞增殖行为的影响  

Effect of sense and antisense VEGF_(165) cDNA transfection on the growth of hypertrophic scar-derived fibroblasts in vitro

作　　者：刘健[1] 王法刚[1] 曹永倩[1] 徐倩[1] 徐荣建[1] 

机构地区：[1]山东大学附属省立医院烧伤整形科,济南250021

出　　处：《山东大学学报（医学版）》2011年第5期135-139,共5页Journal of Shandong University:Health Sciences

基　　金：山东省卫生科技发展计划资助项目(2003HZ109)

摘　　要：目的探讨正、反义血管内皮生长因子(VEGF)基因对人增生性瘢痕成纤维细胞(HHSFb)增殖行为的影响。方法采用组织块法获取HHSFb,行细胞免疫化学鉴定。构建靶向VEGF基因pcDNA3.1(+)/hVEGF165(正义组)、pcDNA3.1(-)/hVEGF165(反义组)和空质粒pcDNA3.1(+)(空载体组)转染人成纤维细胞,以及未行转染处理的人成纤维细胞(对照组),共设为4组。经G418筛选获得阳性转染细胞克隆。逆转录-多聚酶链式反应(RT-PCR)显示VEGF基因在细胞内的表达,上清行酶联免疫吸附反应(ELISA)检测VEGF蛋白表达水平,MTT测定细胞体外生长情况。结果成功构建正、反义VEGF基因表达载体。经统计学分析,与对照组和空载体相比,正义组VEGF mRNA表达增强,蛋白表达增强;反义组VEGF mRNA表达明显减弱,蛋白表达明显降低。MTT结果显示,转染前后细胞体外生长速度基本一致。结论正义VEGF基因重组质粒对HHSFb内源性VEGF的表达有促进作用,反义VEGF基因重组质粒可有效抑制HHSFb内源性VEGF的表达。Objective To explore the effect of sense and antisense vascular endothelial growth factor（VEGF） genes on the growth of hypertrophic scar-derived fibroblasts.Methods Human dermal fibroblasts were isolated from hypertrophic scars by the serum-supplemented media method,and immunocytochemical staining was used to characterize the cell lineage.The genes targeting VEGF including pcDNA3.1（＋）/hVEGF165（the sense group）,pcDNA3.1（-）/hVEGF165（the antisense group） and pcDNA3.1（＋）（the empty vector group） were constructed and transfected into human dermal fibroblasts,and a positive clone was selected by G418.The control group was not transfected.Expression of VEGF mRNA was detected by reverse transcription-PCR（RT-PCR）,and expression of the VEGF protein by enzyme-linked immunosorbent assay（ELISA）.The growth of dermal fibroblasts was measured by MTT.Results The eukaryotic expression plasmids of pcDNA3.1（＋）/hVEGF165 and pcDNA3.1（-）/hVEGF165 targeting VEGF were successfully constructed.Expressions of VEGF mRNA and protein were obviously increased in the sense group,while they were obviously reduced in the antisense group,compared with those in the empty vector group and control group.The growth lines of human dermal fibroblasts before and after transfection were similar.Conclusion The sense VEGF gene could promote VEGF expression in human dermal fibroblasts,while the antisense VEGF gene effectively inhibits this expression.

关 键 词：增生性瘢痕 成纤维细胞 血管内皮生长因子 

分 类 号：R622[医药卫生—整形外科]