The Construction And Identification Of Eukaryotic Vector With A HSC-specific Promoter

AIM: To construct an eukaryotic expression plasmid with a HSC-specific promoter andidentify its specific expression in activated HSCs in vitro.METHODS:①The fragment containing luciferease gene, MCS and polyA tail wasamplifed from PMIR-luciferase using polymerase chain reaction（PCR）. The amplifedproduct was digested with BamH1and Kpn1and cloned into the Bam H1and Kpn1sites inPsmp8plasmid under an αSMA promoter component. The recombinant namedPsmp8+Luciferase+MCS was sequenced to assess the orientation of the insert and thecorrectness of the sequence.②Mouse hepatic stellate cells (HSCs) were isolated fromBALB/C mice’s livers using the way of density gradient centrifugation. Isolated HSCswere activated after being cultured and passaged numbers of days. Expression of αSMAwas determined by western blot and immunofluorescence.③Psmp8+Luciferase+MCS andRenilla luciferase vector were co-transfected HSCs、hepatic cells and kuffer cells. PeGFPplasmid and Renilla luciferase vector were co-transfected the above cells as the negativecontrol. Using Dual-Luciferase Reporter Assay System detected the expression of the twoplasmids in HSCs、hepatic cells、kuffer cells. RESULTS:①The Psmp8+Luciferase+MCS sequence and the orientation of the insertwere sequenced correct.②The cells isolated from mice livers could express αSMAdetermined by western blot and immunofluorescence. So the isolated cells were HSCs andhad been activated.③The Psmp8+Luciferase+MCS could express luciferase in HSCs, butnot in kuffer cells and hepatic cells detected by Dual-Luciferase Reporter Assay System.CONCLUSION: The Psmp8+Luciferase+MCS containing the αSMA promoter couldexpress specifically in activated HSCs. This suggested that Psmp8+Luciferase+MCScontainingαSMA promoter could be used as an specific vector targeted activation HSCs,further more it may be recombined and used in the fibrosis gene therapy.