Construction of Eukaryotic Expression Plasmid of pcDNA3.1/Human Tissue Inhibitor of Metalloproteinase-1-Enhanced Green Fluorescent Protein and Its Expression in Vascular Smooth Muscle Cells

Objective To generate a eukaryotic expression plasmid-pcDNA3.1/human tissue inhibitor of metalloproteinase-1(hTIMP-1)-enhanced green fluorescent protein(EGFP),carrying hTIMP-1 and labeled with EGFP,and to examine the expression of hTIMP-1 in vascular smooth muscle cells(SMCs) transferred with hTIMP.Methods The recombinant plasmids of pcDNA3.1/hTIMP-1-EGFP were obtained by-polymerase chain reaction(PCR) amplification,splicing,and insertion of complementary deoxyribonucleic acid(cDNA) fragments of hTIMP-1 and EGFP.The target gene was transferred to the primarily cultured SMCs(pcDNA3.1/hTIMP-1-EGFP transferred group) by using cationic liposome mediated gene transfection technique.EGFP expression was detected by fluorescence microscopy,and the transfection rate was determined by flow cytometry.Reverse transcriptase polymerase chain reaction(RT-PCR),Western blotting,and other techniques were used to detect the expression of hTIMP-1 gene.The biological activity of matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9(MMP-9) were studied by zymographic analysis of gelatinases.Blank plasmid-pcDNA3.1 transferred SMCs(blank plasmid pcDNA3.1 transferred group) and untransferred SMCs(untransferred group) were used as control.Results In pcDNA3.1/hTIMP-1-EGFP transferred group,the growth ability of SMCs was profoundly inhibited,bright green fluorescence was observed by fluorescence microscopy 24 hours after transfection in SMCs,the rate of transfection analyzed with flow cytometry was 15%,RT-PCR results showed that the genome of hTIMP-1 transferred SMCs contained a 646 bp specific fragment of hTIMP-1 gene,Western blotting results proved hTIMP-1 protein expression in SMCs transferred by hTIMP-1,zymographic analysis of gelatinases showed decreased activity of MMP-2 and MMP-9,compared to those in blank plasmid-pcDNA3.1 transferred group and untransferred group,significant differences were observed(P0.05).Conclusion The generation of a eukaryotic expression plasmid carrying TIMP-1 gene and its expression in SMCs provide a sound basis for hTIMP-1 gene therapy.