Construction of lentiviral vector containing human thrombomodulin gene and its expression in endothelial progenitor cells of rabbits

Abstract

Objective To construct the lentiviral vector containing human thrombomodulin (hTM) gene,and examine the expression of hTM in rabbit peripheral endothelial progenitor cells (EPCs)after transduction and its effect on the biological functions of EPCs.Methods The lentivirus plenti6.3-hTM-IRES-EGFP was reconstructed by polymerase chain reaction (PCR).EPCs were isolated from fresh blood obtained from the heart of a rabbit by density-gradient centrifugation and then were transduced with above lentiviral vector.To evaluate the transduction efficiency of plenti6.3-hTM-IRES-EGFP,quantitativepolymerase chain reaction (Q-PCR),Western blotting and facial action coding system(FACS) were performed.Acetylated low density lipoprotein (DiI-ac-LDL) and fluorescein isothiocyanate (FITC)-UEA-1 double fluorescent labeling was used for the identification of EPCs.Methyl thiazol tetrazolium (MTT) and transwell assays were carried out to examine the proliferation and migration of EPCs in the presence or absence of hTM.Results The recombinant plenti6.3-hTM-IRES-EGFP was confirmed by the evidence of DNA sequence analysis and Western blotting.The transduced EPCs were found overexpressing hTM by Q-PCR,Western blotting and FACS,suggesting the recombinant lentivirus system was successfully constructed.There were no changes in the biological functions of EPCs overexpressing hTM.Conclusion The plenti6.3-hTM-IRES-EGFP has been successfully constructed and hTM can be efficiently and highly expressed in EPCs.All of these provide us experimental evidence for gene-cell combined therapy and further study of TM on inhibiting thrombotic restenosis of arterial occlusive diseases after percutaneous transluminal angioplasty (PTA) treatment. Key words: Thrombomodulin; Lentivirus; Endothelial progenitor cells ; Gene expression