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L Nanoparticles Of Hepatitis B Virus As A Novel Targeted Vector Used For Gene And Drug Therapy Of Hepatocellular Carcinoma

Posted on:2007-03-13Degree:DoctorType:Dissertation
Country:ChinaCandidate:J P CengFull Text:PDF
GTID:1104360212490125Subject:Surgery
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Part 1 Purification and characterization of hepatitis B virus large surface antigen particles produced in Eukaryotic cellObjective Hepatitis B virus (HBV) large surface antigen particles were expressed in Eukaryotic cell using a recombinant plasmid and purified near homogeneity. The particle's physical and biochemical properties were characterized.Methods COS-7 mammalian cell lines were transiently transfected with the recombinant eukaryotic expression vector pSVsigLM~-S~- by the DEAE-Dextran method, Secreted surface protein particles in the media were purified by a strategy based on protein concentration, precipitation and ultracentrifugation. The purity was decided by BCA assay and RIA assay. The molecular size and antigenicity of purified protein was determined by SDS-PAGE and Western blotting. By transmission electron microscopy and atomic force microscopy, the physical properties of sample were characterized.Results L particles were successfully purified from transfected COS-7 cell medium by ultracentrifugation, which purity >90%. L particles consist exclusively of glycosylated 42 kDa proteins which had S and PreS1 antigenicity. HBsAg produced by COS-7 cells had physical properties very similar to 22 nm particles derived from the human plasma and recombinant yeast cells with transmission electron microscopy. But the L particles were observed as large spherical particles with an average diameter of 180 nm by atomic force microscopy in a moist atmosphere.Conclusion L protein can translocate into ER lumen and form subviral particles after budding by fusing a secretion signal sequence to the N terminus of L gene. L particles can be easily purify by ultracentrifugation teclmique and keep its biochemical activity and physical appearance. Part 2 L nanoparticles of HBV as a novel targeted vector used for gene therapy of hepatocellular carcinoma in vitroObjective To investigate the possibility of L nanoparticles of hepatitis B virus as a novel vector used for targeted gene therapy of hepatocellular carcinoma.Methods The GFP expression plasmid was incorporated into L nanoparticles by electroporation. L particles with GFP plasmid were used to tansfect some cell lines from liver parenchyma and cell lines from breast cancer cell and lymphoma. A mixture of L particles and GFP expression plasmid with no electroporation was used as a negative control. On day 2 after transfection, GFP fluorescence was observed by fluorescence microscope.Results Optima] conditions for the weight-based ratio of DNA to L particles in electroporation were determined to be 4:10. Voltage and pulse time was then optimized to 400 V and 60 μs in a similar fashion, respectively. L particles with GFP plasmid yielded higher transfection efficiencies than liposome in human hepatocellular carcinoma cells and normal hepatocytes (P<0.05), whereas human breast cancer MCF-7 cells and human Burkitt's lymphoma cells Raji did not express GFP after transfection with the L particles carrying GFP plasmid. No expression of GFP was observed in the negative controls not subjected to electroporation.Conclusion Electroporation can incorporate plasmid into L particles. L particles can effectively and specific transfect the gene into the human hepatocellular carcinoma cells, which is availability of targeted gene therapy. Part 3 L nanoparticles of HBV as a novel targeted drug delivery system used for drug therapy of hepatocellular carcinomaObjective To investigate the possibility of L nanoparticles of hepatitis B virus as a novel targeted drug delivery system used for drug therapy hepatocellular carcinoma.Methods HepG2 cell lines were incubated with adriamycin in different concentration, meanwhile the mixture of L particles and adriamycin with the same concentration was subjected to electroporation, and then incubated HepG2 cell lines. The cell proliferation and apoptosis rate were deteced by MTT assay and Hoechst staining. The concentration of intercellular ADM was deteced by spectrofluorometer.Results The mixture subjected to electroporation of L particles and adriamycin could significantly inhibit HepG2 cell proliferation in all drug concentration. HepG2 cell lines incubated with the mixture subjected to electroporation had higher apoptosis rate (44.6±3.5%) and intracellular concertration of ADM than with the mixture no subjected to electroporation (20.4±2.7%) and with ADM alone(19.5±2.4%).Conclusion Electroporation can incorporate adriamycin into L particles. The mixture subjected to electroporation of L particles and adriamycin could significantly increase intracellular accumulation of drug, inhabit cell proliferation and induce cell apoptosis, which is availability of targeted delivering drug to liver.
Keywords/Search Tags:Hepatitis B virus, surface antigen, Large protein, Ultracentrifugation, L particles, gene therapy, targeted vector, electroporation, adriamycin, chemotherapy, targeted drug delivery system
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