人肝细胞生长因子基因表达质粒的构建及其活性研究

STUDY ON CONSTRUCTION OF A PLASMID VECTOR CARRYING HUMAN HEPATOCYTE GROWTH FACTOR GENE AND ACTIVITY OF ITS EXPRESSION PRODUCT

目的 :构建一种携带人肝细胞生长因子 (HGF)基因的表达质粒 (pUDKH) ,并对其体外活性进行研究 ,为其体内应用提供依据。方法 :从人胎盘cDNA文库用PCR方法克隆人HGF基因 ,并将其克隆至自行构建的真核表达载体 pUDK上 ,获得表达质粒 pUDKH。将pUDKH体外转染原代培养的骨骼肌细胞 ,分析其转染效率及表达上清中HGF、血管内皮生长因子 (VEGF)的表达水平 ,并采用MTT法分析不同剂量HGF表达产物对人脐静脉内皮细胞的作用。结果 :所克隆构建的携带人HGF基因的质粒 pUDKH可有效转染原代培养的骨骼肌细胞 (0 .0 5 7% ) ,并表达HGF(16～ 18ng/ 4× 10 5cells)和VEGF ,其表达产物对人脐静脉内皮细胞具有明显的增殖刺激活性 (P <0 .0 5 ) ,而且有剂量效应关系。结论 :初步证实本研究构建的质粒

Aim: To construct a plasmid carrying hepatocyte growth factor gene and investigate its effects in vitro . Methods: A complementary DNA(cDNA) clone for human HGF was isolated from human placental cDNA, then subcloned into pUDK vector, which was constructed by ourselves, to form the pUDKH plasmid. The transfection efficiency and the expression level of HGF and VEGF were evaluated by transfecting pUDK or pUDKH into primary rat skeletal muscle cells. The biological effects of HGF expressing product at different doses on endotherial cells were investigated in vitro, and assessed by MTT. Results: The primary rat skeletal muscle cells could be transfected efficiently with pUDKH(0.057%), and secreted HGF(16～18 ng/4×10 5cells) and VEGF proteins. The expressing product could significantly stimulate proliferation of human umibilical vein endotherial cells, in a dose dependent manner( P <0.05).Conclusion: pUDKH has the potential application in vivo to treat ischemic diseases.