碱性成纤维细胞生长因子荧光真核表达载体构建及对过氧化氢诱导血管内皮细胞凋亡的影响

背景：已证实外源性碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）可抑制血管内皮细胞凋亡。目的：构建表达bFGF的荧光真核表达载体,探讨其对过氧化氢（H2O2）诱导的血管内皮细胞凋亡和凋亡相关蛋白的影响。方法：通过基因亚克隆构建荧光真核表达载体pcDNA3.1-bFGF-GFP,利用脂质体介导将bFGF基因导入人脐静脉内皮细胞内,通过荧光观察和RT-PCR检测基因的表达。实验分为3组,对照组（转染pcDNA3.1）、过氧化氢组（转染pcDNA3.1＋H2O2）和bFGF转染＋过氧化氢组（转染pcDNA3.1-bFGF-GFP＋H2O2）,流式细胞术测定细胞凋亡率,Western blot检测caspase-3 P17活性亚单位和Bax蛋白表达。结果与结论：成功构建荧光真核表达载体pcDNA3.1-bFGF-GFP,该载体转染人脐静脉内皮细胞后,bFGF mRNA显著增加,并可观察到绿色荧光。与对照组相比,过氧化氢组细胞凋亡率和caspase-3 P17活性亚单位、Bax蛋白的表达量都明显增加（P〈0.01）,而bFGF转染＋过氧化氢组的细胞凋亡率和caspase-3 P17活性亚单位、Bax蛋白的表达量则比过氧化氢组显著降低（P〈0.01）。证实bFGF基因转染能抑制过氧化氢诱导的血管内皮细胞凋亡,其作用机制可能与调控Bax蛋白表达和caspase-3活性有关。

Construction of basic fibroblast growth factor gene fluorescent eukaryotic cell expression vector and inhibitory effect on hydrogen peroxide-induced apoptosis and related gene expression in vascular endothelial cells

BACKGROUND：It has been verified that exogenous basic fibroblast growth factor（bFGF） has obvious inhibitory effect on apoptosis in vascular endothelial cells.OBJECTIVE：To construct a fluorescent eukaryotic cell expression vector carrying the gene of human bFGF,and to investigate its effect on apoptosis induced by hydrogen peroxide（H2O2） and related gene expression in vascular endothelial cells.METHODS：bFGF and GFP gene was subcloned into eukaryotic expression plasmid pcDNA3.1 by means of gene cloning to obtain pcDNA3.1-bFGF-GFP.pcDNA3.1-bFGF-GFP was transfected into umbilical vein endothelial cells by liposome mediating RT-PCR and observing green fluorescence by fluorescence microscope were used to determine the expression of bFGF and GFP.Umbilical vein endothelial cells were divided into three groups：control group（transfected pcDNA3.1）,H2O2 group（transfected pcDNA3.1＋H2O2） and bFGF-transfected＋H2O2 group（transfected pcDNA3.1-bFGF-GFP＋H2O2）,the apoptosis of umbilical vein endothelial cells was detected by flow cytometry,the expression of caspase-3 P17 subunit and bax protein were determined by Western blot.RESULTS AND CONCLUSION：pcDNA3.1-bFGF-GFP was successfully constructed,bFGF mRNA was increased significantly and specific green fluorescence was observed by fluorescence microscope after pcDNA3.1-bFGF-GFP transfected.Compared with the control group,apoptosis rate,the expression level of caspase-3 P17 subunit and bax protein significantly increased in H2O2 group（P 0.01）.Compared with H2O2 group,bFGF gene transferring significantly decreased apoptosis rate,down-regulated the expression of caspase-3 P17 subunit and bax protein（P 0.01）.bFGF gene transferring can inhibit the apoptosis induced by H2O2 in vascular endothelial cells,the underlying mechanism might be associated with regulation on the expression of bax protein and activity of caspase-3.