重组肝细胞生长因子质粒的构建及对内皮祖细胞增殖的影响

Construction of Recombinant Plasmid of Human Hepatocyte Growth Factor and its Promotes Proliferation of Endothelia Progenitor Cells

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),探讨重组质粒对体外培养的成人外周血中内皮祖细胞(endothelial progenitor cell,EPC)增殖的影响。方法用PCR方法从人胎盘cDNA文库扩增HGF基因,并将其克隆至病毒表达载体pLenti6/V5-Dest,获得表达质粒pLenti-HGF。制备病毒,感染原代培养的内皮祖细胞,免疫组化分析其感染效率及上清中HGF的表达水平。MTT法检测感染病毒后对细胞增殖的影响。结果 pLenti-HGF病毒可有效感染体外培养的内皮祖细胞,表达HGF,并能促进内皮祖细胞的增殖。结论内皮祖细胞经病毒感染后可以表达hHGF,外源性hHGF基因能够刺激内皮祖细胞增殖。

Objective To investigate the effect of the recombinant human hepatocyte growth factor（HGF） on proliferation of endothelial progenitor cells in vitro.Methods The full length cDNA of hHGF was amplified from human placenta cDNA library by polymerase chain reaction（PCR）,and was cloned into pLenti6/V5-Dest virus expression plasmid.The virus was used to infect the cultured endothelial progenitor cells.Expression of hHGF in the endothelial progenitor cells were measured by immunohistochemical staining and ELISA.The effect of HGF on endothelial progenitor cells proliferation was measured by MTT colorimetric assay.Results Endothelial progenitor cells were effectively infected by pLenti-HGF virus in vitro.Human HGF protein was detected by the immunohistochemical staining.pLenti-HGF virus infection had improved the proliferation of endothelial progenitor cells.Conclusion Recombinant hHGF can stimulate the proliferation of endothelial progenitor cells.