Objective: To study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localiza...Objective: To study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells. Methods: Seven truncated CORE-GFP (green fluorescent protein) fusion protein expression plasmids were constructed, which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1?172 aa, 1?126 aa, 1?58 aa, 59?126 aa, 127?172 aa; BNT: 1?172 aa and C191: 1?172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry. Results: Different truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1?172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT>BNT>C191). To the different fragment COREs of BT, N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1?172 aa>1?126 aa>1?58 aa>127?172 aa>59?126 aa). Conclusion: These results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of dif- ferent HCV quasi-species might have some difference in their pathogenesis.展开更多
The aim of this study was to compare six methods of detecting apoptosis induced by extracellular adenosine triphosphate (ATP) in human leukemic lymphocytes with purinergic P2Z receptors.These...The aim of this study was to compare six methods of detecting apoptosis induced by extracellular adenosine triphosphate (ATP) in human leukemic lymphocytes with purinergic P2Z receptors.These methods used were electron microscopy(EM), detection of internucleosomal DNA fragmentation by agarose gel electrophoresis, autoradiographic analysis of DNA fragmentation, in situ labeling of DNA strand breaks with fluorescein dUTP and exogenous terminal deoxynucleotidyl transferase (TUNEL), quantitation of 3 ends of DNA breaks by labeling with α 32 PdCTP(TdT assay), and quantitation of apoptotic cells with fluorescein annexin V using flow cytometry(FCA).We found EM and detection of DNA ladder pattern by agarose gel electrophoresis to be specific,but lacking in sensitivity. The combination of autoradiography and gel electrophoresis gave an increase in sensitivity of at least 50 fold although, of all the methods, the TdT assay was shown to be most sensitive. The four methods for quantifying apoptosis EM, FCA, TUNEL and TdT assay proved to be reliable and gave statistically similar results on apoptotic lymphocytes. These observations indicate it is essential to combine specific, sensitive and quantitative techniques in detecting apoptosis.展开更多
基金Project (No. 2001BA705B06) supported by a grant from the National Key Technologies R&D program (10th Five-Year Plan) of China
文摘Objective: To study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells. Methods: Seven truncated CORE-GFP (green fluorescent protein) fusion protein expression plasmids were constructed, which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1?172 aa, 1?126 aa, 1?58 aa, 59?126 aa, 127?172 aa; BNT: 1?172 aa and C191: 1?172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry. Results: Different truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1?172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT>BNT>C191). To the different fragment COREs of BT, N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1?172 aa>1?126 aa>1?58 aa>127?172 aa>59?126 aa). Conclusion: These results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of dif- ferent HCV quasi-species might have some difference in their pathogenesis.
文摘The aim of this study was to compare six methods of detecting apoptosis induced by extracellular adenosine triphosphate (ATP) in human leukemic lymphocytes with purinergic P2Z receptors.These methods used were electron microscopy(EM), detection of internucleosomal DNA fragmentation by agarose gel electrophoresis, autoradiographic analysis of DNA fragmentation, in situ labeling of DNA strand breaks with fluorescein dUTP and exogenous terminal deoxynucleotidyl transferase (TUNEL), quantitation of 3 ends of DNA breaks by labeling with α 32 PdCTP(TdT assay), and quantitation of apoptotic cells with fluorescein annexin V using flow cytometry(FCA).We found EM and detection of DNA ladder pattern by agarose gel electrophoresis to be specific,but lacking in sensitivity. The combination of autoradiography and gel electrophoresis gave an increase in sensitivity of at least 50 fold although, of all the methods, the TdT assay was shown to be most sensitive. The four methods for quantifying apoptosis EM, FCA, TUNEL and TdT assay proved to be reliable and gave statistically similar results on apoptotic lymphocytes. These observations indicate it is essential to combine specific, sensitive and quantitative techniques in detecting apoptosis.
