| [Background and Objective]Hepatic fibrosis which is characterized by the activation and proliferation of hepatic stellate cell (HSC), associated with the production and deposition of extracellular matrix (ECM) in the Disse's space is a pathological response to various chronic liver diseases. As successful approaches to gene therapy on hepatic fibrosis, repressing activation and proliferation of HSC, promoting the degradation of ECM as well as improving liver regeneration are focused on. Therefore, it has been considered that the targeted delivery of genes which could promote degradation of ECM and liver cell proliferation would favor reversal of hepatic fibrosis and provide a new tool for gene therapy of hepatic fibrosis.Urokinase-type plasminogen activator (uPA) is a kind of glucoprotein which can regulate matrix remodeling in hepatic fibrosis. Many observations have revealed that uPA acts to generate plasmin which is a broad-spectrum proteinase capable of directly degrading matrix components. It is more important that plasmin also participates in matrix degradation inderectly by activating the matrix metalloproteinases (MMPs) which are a family of zinc-dependent endopeptidases secreted in latent inactive forms. The activated MMPs are capable of degrading most matrix components. Many studies have indicated that absence of uPA would result in accelerated fibrosis in liver, lung and other organs.Hepatocyte growth factor (HGF) was first recognized as a molecule that stimulates hepatocyte proliferation. The active form is a heterodimer of a heavy a chain (69 kDa) and a light chain (34 kDa), which results from proteolytic cleavage of the precursor. HGF binds to hepatocytes via c-met/HGF receptor and promotes cell proliferation. In addition, some results showed that HGF gene therapy could improve the liver cirrhosis by inhibiting TGF- expression.Recombinant adenoviruses are currently used for gene transfer in vitro and gene therapy. Several features of adenovirus biology have been made as vectors of choice for these applications. For example, adenoviruses can transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, hightiters of viruses and high levels of transgene expression generally can be obtained. AdEasy?system has more advantages over the other methods in constructing recombinant adenoviruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria based on AdEasy?system rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral porduction is conveniently followed with the aid of green fluorescent protein (GFP), encoded by a gene incorporated into the viral backbone, which can be allowed directly to observe the efficiency of transfection and infection. Homogeneous viruses can be obtained from this procedure without plaque purification. All of these can significantly decrease the time required to generate viruses.The purpose of this study was designed to construct two kinds of replication-deficient recombinant adenoviruses by AdEasy?system rapidly: AdHGF-inserted with HGF cDNA; AduPA-inserted with non-secreted human uPA cDNA, then both of them would be used for gene transfer in hepatocyte or HSC and the combinable genes delivery in experimental hepatic fibrosis, which could provide a new pathway for the combination of gene therapy by multiple genes delivery in hepatic fibrosis.[Methods]The HGF and uPA cDNA were obtained from the plasmids by digestion or PCR amplification, and uPA cDNA was modified by RR-signal and KDEL-signal, which would keep the uPA protein retained within the cells and avoid the risk of hemorrhage secondary to uPA secretion. The shuttle plasmids- pAdTrack-CMV-HGF and pAdTrack-CMV-uPA were established by ligation respectively. Then the linearized shuttle plasmids were co-transformed into colibacillus BJ5183 with backbone vector AdEasy-1 to obtain the recombinant adeno...
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