ABSTRACT
<p><b>OBJECTIVE</b>To study the cytotoxic T lymphocyte (CTL) response induced by dendritic cells (DC) transduced with recombinant adenovirus vector bearing hepatitis B virus surface antigen (HBsAg) gene in hepatocellular carcinoma HepG2. 2. 15 cells in vitro.</p><p><b>METHODS</b>Full length HBsAg cDNAs were subcloned into pIND vector, followed by being cloned into pShuttle vector. The HBsAg gene fragments resulted from the pShuttle-S digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA. After packaged with HEK293 cells, the adenovirus expression vector was obtained. Then the recombinant adenovirus expression plasmid AdVHBsAg was transfected into human monocyte-derived dendritic cells, to construct AdVHBsAg hepatocarcinoma tumor vaccine. The effectiveness of transfection was detected by Western blot. Surface molecules of AdVHBsAg-DC were detected by FACS. Autologous T cell proliferation stimulated by AdVHBsAg-DC was detected by 3H-TdR assay. Cytotoxic CTL activity induced by AdVHBsAg-DC in vitro was detected by LDH assay.</p><p><b>RESULTS</b>HBsAg gene in the inserted DNA of AdVHBsAg was confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. Cell pathological changes appear after 10 days HEK293 cells transfected AdVHBsAg. Western blot analysis showed that HBV surface antigen gene was expressed in transfected DC, indicating that the transfection was effective. AdVHBsAg-DC was able to upregulate CD1a, CD11c, CD80, CD86 and HLA-DR. Autologus T cell proliferation induced by AdVHBsAg-DCs was significantly higher than that in DC control group and LacZ-DC group (P < 0.05). AdVHBsAg-DC activated CTL presented the specific killer ability to the hepatocellular carcinoma cells expressing HBsAg.</p><p><b>CONCLUSION</b>DC transduced with recombinant adenovirus HBsAg can express HBV-related hepatocellular carcinoma antigen (HBsAg), and AdVHBsAg-DC can induce potent immune response against HBsAg-positive hepatocellular carcinoma cells in vitro.</p>
Subject(s)
Humans , Adenoviridae , Genetics , Antigens, CD1 , Metabolism , CD11c Antigen , Metabolism , Cancer Vaccines , Allergy and Immunology , Carcinoma, Hepatocellular , Allergy and Immunology , Pathology , Virology , Cell Line, Tumor , Cell Proliferation , Cytotoxicity, Immunologic , Allergy and Immunology , Dendritic Cells , Cell Biology , Allergy and Immunology , Metabolism , Genetic Vectors , Hepatitis B Surface Antigens , Genetics , Metabolism , Liver Neoplasms , Allergy and Immunology , Pathology , Virology , Plasmids , Recombinant Proteins , Genetics , Metabolism , T-Lymphocytes, Cytotoxic , Cell Biology , Allergy and Immunology , TransfectionABSTRACT
<p><b>AIM</b>To study the biliary excretion of genistein and its metabolite at different doses in rats.</p><p><b>METHODS</b>Suspended in 0.5% CMC-Na solution, genistein was orally administered to rats at the dose of 6.25, 12.5 and 50 mg x kg(-1), separately. At various time intervals, the bile was collected. The bile was treated with beta-glucuronidase. The genistein in bile was extracted twice by vortexing with 2.0 mL mixture of methyl tert-tubtyl ether and pentane (8:2). The organic phase was removed into the tubes and then evaporated in ventilation cabinet. The residue was dissolved in 50 microL of methanol. Twenty microL solution was drawn and detected by high-performance liquid chromatography.</p><p><b>RESULTS</b>The accumulative biliary excretion of genistein was (42.56 +/- 6.54) , (75.17 +/- 18.87) and (126.60 +/- 34.78) microg at the dose of 6.25, 12.5 and 50 mg x kg(-1), respectively. The total drug (genistein plus glucuronidated genistein) excreted from bile was (108.46 +/- 35.23), (423.46 +/- 158.31) and ( 853.74 +/- 320. 84) microg, and the ratio of glucuronidated genistein was 60.76% , 82.25% and 85.17% at the dose of 6.25, 12.5 and 50 mg x kg(-1), respectively.</p><p><b>CONCLUSION</b>The genistein was excreted mainly in the form of glucuronidated genistein in rat bile. The genistein and glucuronidated genistein were excreted in a nonlinear dose-dependent manner.</p>
Subject(s)
Animals , Female , Male , Rats , Administration, Oral , Bile , Metabolism , Dose-Response Relationship, Drug , Genistein , Chemistry , Metabolism , Pharmacokinetics , Molecular Structure , Phytoestrogens , Metabolism , Pharmacokinetics , Rats, Sprague-DawleyABSTRACT
This article reviews the structure, physical and chemical characteristics of bone morphogenetic protein, summarized the effects of bone morphogenetic protein on hematopoiesis in embryo and adult animal, discussed the possible mechanisms and pointed out the theoretic and practical significance on this research work.