Study On Hepatocyte-tageted Gene Transduction Of Chitosan In Vivo

Gene therapy is one of the hot issues of modern medical research work. Hepatocyte-targeted gene therapy is attached more and more importance. In recent years, hepatocyte-targeted gene therapy made a rapid progress. Hepatocyte is an ideal target site of directional gene transduction in that liver is one of organs that communicate the blood directly. Furthermore, the endothelial cell of hepatic sinusoid has little connective tissue. There are many windows without membrane structure which vary from 0.1 micrometer to 1 in the diameter. Therefore, the blood vascular system of liver has a loose permeability. Some macromolecule substance can pass through freely. There is no exact barrier structure between blood and liver.With the limit of viral vector's insecurity, present gene therapy are mainly about non-viral vector. The traditional non-viral vector has lowertransfection efficacy. Chitosan is a new type of nanoparticles vector. It is biodegradable and biocompatible. It has high security and transfection efficacy. The studies of others indicate that chitosan has high transfection efficacy in vitro, so do mice in vivo. But with regard to gene transduction in large animals, there is no reference internationally.In this study, we choose Galactosylated chitosan (GC). It contains galacose ligand which can bind to the asialoglycoprotein receptor (ASGP-R) on the surface of hepatocyte. In this way comes to the hepatocyte-targeted gene therapy.Objective: To investigate the gene transfer efficacy of galactosylated chitosan (GC) in vitro and in vivo, and the hepatocyte-targeted of GC in large animals(canis). Then investigate the possibility of hepatocyte-targeted gene therapy mediated by GC.METHODS: Extract plasmids pEGFP-N1 and pBIND which contain report gene in large amount. GC was complexed with plasmids DNA to develop nanoparticle complex. Then transfect them to SMMC-7721 cell in culture which compare to the liposome transfection reagent Lipofectamine? 2000. After 48 hours, observe the expression of report gene and the transfection efficacy. canis was treated with an intravenous injection of gadolinium chloride (14mg/kg) 24 hours before operation in order to depress Kupffer cells. The nanoparticles which contained 1mg plasmid were given through hepatic artery and portal vein. Harvest the main organs and make frozen section after 48 hours. Observe whether there was green fluorescence under the fluorescence microscope. With naked plasmid DNA as control.RESULTS: Green fluorescent protein was detected in SMMC-7721...