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Expression of nuclear factor kappa B in hepatitis C virus core gene transfected cholangiocarcinoma cells

Chinese Medical Journal 2002年第7期115卷 998-1001,145页

作者：刘小方邹声泉裘法祖华中科技大学同济医学院附属同济医院普外科武汉430030

OBJECTIVE: To establish an experimental model for exploring the role of hepatitis C virus (HCV) in the development of cholangiocarcinoma. METHODS: Recombinant plasmids of HCV-C gene were constructed by molecular cloning techniques and identified by PCR and restriction enzyme *** plasmids were then transfected into QBC939 cells (a cholangiocarcinoma cell line) by Lipofection. After selection with G418, resistant colonies were obtained and analyzed by immunocytochemistry and Western blotting. The morphology was observed by trans mission electron microscopy (TEM). The expression of NF-(k)B was detected by immunocytochemistry. RESULTS: Recombinant plasmid was shown by PCR and restriction enzyme mapping to carry the target gene. Moreover, it could efficiently express HCV-C protein in QBC939 cells. HCV-like particles were found in the cytoplasm by TEM, which were spherical with a diameter of 50-80 nm and possessed an outer membrane. Moreover, NF-(k)B activation could be shown in HCV core-transfected cells. CONCLUSION: Expression of the HCV-C gene in cholangiocarcinoma cells was achieved. Transfected tumor cells (QBC939-HCVc) could be used as a model to study the effect of HCV on the development of cholangiocarcinoma.

关键词： Bile Ducts, Intrahepatic Bile Duct Neoplasms Cholangiocarcinoma Humans NF-kappa B Plasmids Polymerase Chain Reaction Transfection Tumor Cells, Cultured viral core proteins

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Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

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World Journal of Gastroenterology 2001年第3期7卷 370-375页

作者： Ji Lin Cheng Bao Ling Liu Yi Zhang Wen Bin Tong Zheng Yan Bai Fang Feng Institute of Hepatology,Peoples Hospital,Medical Center of Beijing University,Beijing 10(X)44,China Institute of Hepatology People's Hospital Medical Center of Beijing University of Beijing China.

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

关键词： B-Lymphocytes Cells, Cultured Female Hepacivirus development purification Herpesvirus 4, Human Humans Immunohistochemistry In Vitro Polymerase Chain Reaction RNA, viral Research Support, Non-U.S. Gov't Reverse Transcriptase Polymerase Chain Reaction Transformation, Genetic viral core proteins viral Nonstructural proteins Virus Replication

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Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

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World Journal of Gastroenterology 2001年第4期7卷 583-586页

作者： Hong-Chao Zhou De-Zhong Xu Xue-Ping Wang Jing-Xia Zhang Ying-Huang Yong-Ping Yan Yong Zhu Bo-Quan Jin Department of Epidemiology,the Fourth Military Medical University,Xi’an 710033,Shaanxi Province,ChinaDepartment of Immunology,the Fourth Military Medical University,Xi’an 710033,Shaanxi Province,China Department of Immunology the Fourth Military Medical University Xi'an 710033 Shaanxi Province China. juliu@***

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide "ALAHGVRAL (core 150-158)". The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.

关键词： Amino Acid Sequence Antibodies, viral B-Lymphocytes Cell Line Epitope Mapping HLA-A2 Antigen Hepacivirus Hepatitis C Humans Peptide Fragments Predictive Value of Tests Research Support, Non-U.S. Gov't T-Lymphocytes, Cytotoxic viral core proteins

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