肝细胞生长因子基因转染对人脐静脉内皮细胞增殖、迁移的影响

摘要

Objective:To investigate the effects of hepatocyte growth factor(HGF)gene on the proliferation and migration of human aumbilicalvein endothelial cells(ECV 304).Methods:The plasmid pRc/CMV-HGF was used as the template to amplify HGF gene by PCR.The products were cloned in eukaryotic expression vector pEGFP-N1 to construct recombinant plasmids as pEGFP-HGF.The recombinaint plasmids pEGFP-HGF were identified by restrictive endonuclease digestion and sequencing,then transfected into human umbilical vein endothelial cellswith liposome,and positive clones were selectited by G418 .The transfection and the expression of HGF IN ECV 304 were tested by PT-PCR and immunohistochemistry.ELISA was performed to detected levels of HGF in ECV 304.Proliferative activites of the cells were evaluated by MTT assay.Transwellmigration assay was performed to detectmigration activity.Results:The recombinaintplasmids were qualified by restrictive endonuclease digestion and squencing.After G418 selection,the cell clones were obtained succesfully.Under a fluorescent microscope,the expression of enhanced green fluorescent protein(EGFP)was seen in cells transfected with vector contain EGFP gene.RT-PCR and immunohistochemistry proved that there was transcription of HGF gene in transfection cells and there was expression of HGF in these cells.The HGF levels in the HGF/ECV 304 cells increased significantly at the 48 th hour after thansfection.MTT and transwellmigration assay showed HGF promoteproliferation and migration of the cultured human umbilicalvein endothelial cells.Conclusion:The recombinant of pEGFP-HGF is an effective expression vector.HGF produced by the transfected cells promotes the proliferation and migration of the human umbilicalvein endothelial cells,HGF is thus a potent agent for strategies designed to promote therapeutic angiogenesis.%目的:观察HGF基因对人脐静脉内皮细胞增殖、迁移的影响.方法:从已有质粒pRc/CMV-HGF中扩增出HGF基因,将其克隆到含增强型绿色荧光蛋白的真核表达载体中,构建重组质粒pEGFP-HGF,酶切及测序鉴定正确后,用脂质体将重组质粒pEGFP-HGF转染到人脐静脉细胞株ECV304中,G418筛选获得稳定表达细胞克隆,采用荧光显微镜观察、RT-PCR、免疫细胞化学方法检测鉴定重组质粒的表达情况;酶联免疫吸附法(ELISA)检测稳定表达细胞中HGF 的含量;再以MTT法检测转染重组质粒后细胞增殖的改变,Transwell Migration实验检测细胞迁移能力的改变.结果:所构建重组质粒经酶切图谱分析和序列测定证实构建成功;荧光显微镜下观察到有绿色荧光;RT-PCR证实 HGFmRNA在转染阳性细胞高表达;免疫细胞化学证实转染pEGFP-HGF质粒的细胞有HGF蛋白的表达;ELISA检测细胞培养基中HGF含量可达112.3 ng/ml,MTT法、Transwell Migration测定转染pEGFP-HGF阳性细胞的增殖、迁移能力明显高于对照组(P＜0.01).结论:重组质粒pEGFP-HGF能够在内皮细胞株ECV304转录、表达;表达的活性蛋白HGF刺激ECV304的增殖、迁移,为进一步应用于基因治疗奠定实验基础.