摘要
目的 :研究牛bFGF -pcDNA3 在体外培养人脐静脉内皮细胞中的表达与活性 ,及对该细胞生物学特性的影响。方法 :脂质体法将bFGF -pcDNA3 真核表达载体转染体外培养的人脐静脉内皮细胞 ,经G4 18筛选阳性克隆。通过免疫细胞化学法检测bFGF的表达部位及Ⅷ因子相关抗原的表达。应用化学发光Westernblotting法检测bFGF在转染细胞与未转染细胞中的表达。绘制细胞的生长曲线并计算倍增时间 ,采用MTT法对bFGF的生物活性进行估计。结果 :bFGF -pcDNA3 转染人脐静脉内皮细胞后 ,细胞呈梭形者增多。免疫细胞化学证实转染前后细胞均为血管内皮来源细胞 ,转染与未转染细胞均在胞浆内表达bFGF。Westernblotting证实细胞转染前后均表达 17kDbFGF ,但转染后表达量增高 ,细胞生长曲线上移 ,倍增时间缩短。MTT法证实转染细胞产生的bFGF其生物学活性约相当于持续添加外源性bFGF 5 71 4ng/L。结论 :转染bFGFcDNA的人脐静脉内皮细胞能在体外增殖 ,并具有表达与分泌活性bFGF的功能。bFGF -pcDNA3 可引起内源性bFGF表达增高 ,对血管内皮细胞产生相应的生物学活性。
AIM: To study the expression and activity of bovine bFGF-pcDNA_3 in cultured human umbilical vein endothelial cells and its effect on the bioactivity of these cells. METHODS: Recombinant bovine bFGF-pcDNA_3 was transferred into cultured human umbilical vein endothelial cells by lipofectin transfection, and positive clones were selected by G418. The bFGF expression position and Ⅷ related antigen was examined by immunohistochemical analysis. Chemiluminescence Western blotting analysis of total cell extracts was carried to detect bFGF expression in transfected and non-transfected cells and to compare their expression level. Growth curves of transfected and non-transfected cells were drawn and doubling time were caculated. The activity of bFGF was estimated by MTT analysis. RESULTS: Human umbilical vein endothelial cells appeared elongated after transfection. Immunohistochemical analysis confirmed its endothelial origin both before and after transfection and demonstrated that both transfected and non-transfected cells express bFGF in cytoplasm. Western blotting confirmed that both cell lines express 17kD bFGF but the level is much higher in transfected cells. Growth curve of transfected cell line moves upward and the doubling time is shorter. MTT analysis confirmed that the bioactivity of the expression product is equal to about 571.4 ng/L of exogenous bFGF continuous stimulation. CONCLUSION: bFGF cDNA-transfected human umbilical vein endothelial cells can grow in vitro and express functional bFGF. bFGF-pcDNA_3 can increase endogenous bFGF expression and has corresponding bioactivity on human umbilical endothelial cells.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2002年第10期1228-1232,T001,共6页
Chinese Journal of Pathophysiology
关键词
成纤维细胞生长因子
碱性
脐静脉
内皮细胞
Fibroblast growth factor, basic
Umbilical veins
Endothelial cell
