Construction Of Hepatocyte High Expression System Of Vectors Driven By HBV Gene Promoter And Study Of Anticancer Effect

Hepatocellular carcinoma(HCC) is one of the most common maliganant tumors in the world, responsible for an estimated one million deaths annually. The prognosis of HCC patients is generally very poor despite many treatment strategies. Gene therapy is an exciting approach to treat HCC in the biological and clinical research. The success of gene therapy largely depends on the development of a vector or vehicle that can selectively and efficiently deliver a gene to tumor cells or express therapeutic gene only in tumor tissues with minimal toxicity in normal hepatic cells or nonhepatic cells. Study of the molecular virology revealed the mechanism of the expression and regulation of HBV, which may attribute to the construction of hepatic-specific expression system of vector. HBV has a partially double-stranded 3.2-kb DNA genome, and all the regulating sequence located in the protein encoding region. Four promoters(Cp, SP I, SPII, Xp) have been identified as cis regulators for transcription of the 3.5-, 2.4-, 2.1-and 0.8-kb mRNAs, respectively. Two regions(nt1074 to 1234, nt1627 to 1774) in the HBV genome have been shown to act as transcriptional enhancers, Enhancer I (EN I )and Enhancer II(EN II), which show highly conservative trait, A lot of hepatocyte-specific factors bind to those two regions. For instance, C/EBP, HNF-4 and HB1F bind to EN I region, and the transcriptional factors, like C/EBP, HNF-4, HLF, HNF3, FTF and E4BP4, bind to EN II region. Only in the hepatocyte cells can these transcriptional factors be detected, but not in nonhepatic cells. This suggests that these transcriptional factors are necessary for the hepatocyte-specific traits of the EN I and EN II, and the hepatotropism of HBV replication might be related with these transcriptional factors.Therefore we use the transcriptional regulatory sequence which might be attributable to the hepatotropism of HBV and use the therapic genes which only inhibit or kill tumor cells to construct the hepatic gene therapy vectors. When transferred into body, they will kill the hepatic tumor cells without damaging the normal cells. So we carry out our studies as follows: (1) Cloning of different HBV promoters and assessing of transcriptional activity of HBV promoter in hepatocyteor nonhepatic cells. (2) Construction of apoptin, antisense hTRT, IL-24 andoncostatin M(osm) eukaryotic expression vectors driven by the HBV promoter and study of its expression specificity and anticancer effect in vitro and in vivo.The preliminary research results as follows:I. Construction of hepatocyte-specific vectors1. Five Luciferase reporter plasmids driven by HBV EN II with basic core promoter(BCP), EN I and EN II with BCP, HBV EN II and BCP with mCMV promoter were successfully constructed.2. A high hepatocyte-specific expression vector was achievedThe recombinant plasmids were transfercted into five hepatic cells(HepG2, 2.2.15, BEL-7402, SMMC7721, LO2) and five nonhepatic cells(HeLa, H460, MCF7, 803, A375)using Lipofectamine?reagent, The Luciferase expression which indirectly represented the transcriptional activeity of HBV promoter lying upstream of Luc gene was detected with Dual-Luciferase Reporter Assay System. the SV40 enhancer/promoter (pGL3-Control) as a positive control, and the negative control without promoter (pGL3-Basic). The Luciferase activity of pGL3-Control plasmid in each cell line was considered as 100%. The HBV promoter construct showed significant transcriptional activity in all hepatic cell lines., the transcriptional activity of EN IIR (mutant type) promoter was 0.987-5.79 fold of positive control under SV40 promoter in hepatic cell lines; the transcriptional activity of EN I -IlW(ayw type) promoter was 1.25-15.31 fold of positive control under SV40 promoter in hepatic cell lines; the transcriptional activity of EN I -IIR promoter was 3.76-21.83 fold of positive control under SV40 promoter in hepatic cell lines; the transcriptional activity of ENIImCMV promoter was 9.49-91.71 fold of positive control under SV40 promoter in hepatic cell lines...