AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was ap...AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.展开更多
INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-...INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.展开更多
AIM: To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation.METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promot...AIM: To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation.METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control.To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CDPCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to10 μmol/L and mutual primer to about 100 nmol/L. Optimized CD-PCR could detect both mutant and wild strain indepe ndent of the amount of templates and the number of PCRcycles. Its detection limit was 103 copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.展开更多
INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For...INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).展开更多
AIM:The association of hepatitis C virus(HCV) infection with typeⅡmixed cryoglobulinemia is well established,but the role of HCV in B-cell lymphoma remains controversial.In patients with HCV infection,B-cell clonal e...AIM:The association of hepatitis C virus(HCV) infection with typeⅡmixed cryoglobulinemia is well established,but the role of HCV in B-cell lymphoma remains controversial.In patients with HCV infection,B-cell clonal expansions have been detected in peripheral blood and bone marrow,and a high prevalence of B-cell non-Hodgkin's lymphomas has been documented.Liver biopsies in chronic HCV infection frequently show portal lymphoid infiltrates with features of B follicles,whose clonality has not yet been investigated.The object of this study was to determine the frequency of liver-infiltrating monoclonal B-cells in 40 patients with HCV infection.METHODS:Eight hundred and forty-eight patients were studied prospectively,including 40 HCV-positive patients and 808 patients with chronic hepatitis B virus(HBV)infection.Immunohistochemical study for B-and T-cell markers was performed on the paraffin-embedded liver tissue sections.The clonality of lymphoid B-cells was tested using a polymerase chain reaction(PCR)approach designed to identify immunoglobulin heavy chain gene(IgH) rearrangements.RESULTS:Liver-infiltrating monoclonal B-cells were detected in the liver for 4(10%)of 40 HCV-positive patients but were present in only 3(0.37%)of 808 liver biopsy specimens with chronic HBV infection.Chi-square testing showed that the monoclonal B-cells infiltration in the liver was more frequent in the HCV-infected patients(P=0.000).A clonal IgH rearrangement was detected in 5(71.4%)of 7 liver biopsy specimens with monoclonal B-cells infiltration.In 2 of 5 patients with both a clonal B-cell expansion and monoclonal B-cells infiltration in the liver,a definite B-cell malignancy was finally diagnosed.CONCLUSION:Liver-infiltrating monoclonal B-cells are detected in the liver of patients with chronic HCV and HBV infection.A high percentage of patients with monoclonal B-cells infiltration and B-cell clonality in the liver were finally diagnosed as having a definite B-cell malignancy.展开更多
INTRODUCTION Hepatitis B is one of the common infectious diseases,which severely impairs the health of the people in our country and has close relationship
INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivat...INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes展开更多
To develop a new method for detecting the A(83) point mutation in HBV preC region, a mispairing primer was designed, which could introduce a Bsu 36I restriction site into the amplified product of A(83) mutant. After p...To develop a new method for detecting the A(83) point mutation in HBV preC region, a mispairing primer was designed, which could introduce a Bsu 36I restriction site into the amplified product of A(83) mutant. After polymerase chain reaction(PCR), the amp展开更多
Aim The 3’-base specific polymerase chain reaction (3’- BS- PCR) method was es-tablished to investigate the relationship between the mutation of precore region of Hepatitis B virus(HBV) and the liver damage to the p...Aim The 3’-base specific polymerase chain reaction (3’- BS- PCR) method was es-tablished to investigate the relationship between the mutation of precore region of Hepatitis B virus(HBV) and the liver damage to the patients caused by HBV and the possibility of HBV precore gene in-tegration in liver cells。 Mdthods According to the DNA sequence of precore region of HBV,themethod of 3’- BS- PCR is applied to analyze the point mutation site 1896 of HBV precore in 126 clini-cal serum specimens and 23 hepatoeellular carcinoma (HCC) patients’ tissues and serum whose trmorshave been surgically excised and pathologically diagnosed.Rdsults The point mutation in site 1896 ofHBV precore has been successfully rates of preore gene of HBV in the 23 patients’ tissues and serum are52.2 % (12/23) and 30.4 % (7/23) respectively.Conclusion The established method for HBV ore-core mutation analysis is simple and results can well repeated.It has provided a new approach to clinicalHBV research and its relationship to liver damage.The results obtained suggested that HBV precoremutation exists in a wide range among serum and tissue of the patients infected by HBV and HCC pa-tients,and the pre-c gene of HBV can not be detected in the serum of 21.8% of the HCC patients(tissue HBV precore gene positive).We may deduce that there may be the integration of HBV precoregenee in the genome of liver cells,which may play an important role in the carcinogenesis of HCC.展开更多
基金Supported by a National Research Foundation(NRF)of Korea grant funded by the Korean government(Ministry of EducationScience+2 种基金and TechnologyMEST)Grant No.2013-005810
文摘AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.
文摘INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.
基金Supported by the Science Foundation of Guangdong Province,No.99M04801G
文摘AIM: To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation.METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control.To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CDPCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to10 μmol/L and mutual primer to about 100 nmol/L. Optimized CD-PCR could detect both mutant and wild strain indepe ndent of the amount of templates and the number of PCRcycles. Its detection limit was 103 copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.
基金This work was supported by Projects of Tackling Key Problems in ScienceTechnology from the State Science+2 种基金Technology Ministry (TJ99-LA01) Shanghai ScienceTechnology Commission (994919033 )
文摘INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).
基金Supported by National Natural Science Foundation of China,30271181
文摘AIM:The association of hepatitis C virus(HCV) infection with typeⅡmixed cryoglobulinemia is well established,but the role of HCV in B-cell lymphoma remains controversial.In patients with HCV infection,B-cell clonal expansions have been detected in peripheral blood and bone marrow,and a high prevalence of B-cell non-Hodgkin's lymphomas has been documented.Liver biopsies in chronic HCV infection frequently show portal lymphoid infiltrates with features of B follicles,whose clonality has not yet been investigated.The object of this study was to determine the frequency of liver-infiltrating monoclonal B-cells in 40 patients with HCV infection.METHODS:Eight hundred and forty-eight patients were studied prospectively,including 40 HCV-positive patients and 808 patients with chronic hepatitis B virus(HBV)infection.Immunohistochemical study for B-and T-cell markers was performed on the paraffin-embedded liver tissue sections.The clonality of lymphoid B-cells was tested using a polymerase chain reaction(PCR)approach designed to identify immunoglobulin heavy chain gene(IgH) rearrangements.RESULTS:Liver-infiltrating monoclonal B-cells were detected in the liver for 4(10%)of 40 HCV-positive patients but were present in only 3(0.37%)of 808 liver biopsy specimens with chronic HBV infection.Chi-square testing showed that the monoclonal B-cells infiltration in the liver was more frequent in the HCV-infected patients(P=0.000).A clonal IgH rearrangement was detected in 5(71.4%)of 7 liver biopsy specimens with monoclonal B-cells infiltration.In 2 of 5 patients with both a clonal B-cell expansion and monoclonal B-cells infiltration in the liver,a definite B-cell malignancy was finally diagnosed.CONCLUSION:Liver-infiltrating monoclonal B-cells are detected in the liver of patients with chronic HCV and HBV infection.A high percentage of patients with monoclonal B-cells infiltration and B-cell clonality in the liver were finally diagnosed as having a definite B-cell malignancy.
基金the Natural Science Foundation of Anhui Province,No.9741006Natural Science Foundation of Anhui Educational Commission,No.JL-97-077.
文摘INTRODUCTION Hepatitis B is one of the common infectious diseases,which severely impairs the health of the people in our country and has close relationship
基金Projects of the Science Development Foundation of Shanghai (994919033)Tackling Key Problems in Science and Technology from the State Science and Technology Ministry(TJ99LA01)
文摘INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes
文摘To develop a new method for detecting the A(83) point mutation in HBV preC region, a mispairing primer was designed, which could introduce a Bsu 36I restriction site into the amplified product of A(83) mutant. After polymerase chain reaction(PCR), the amp
基金Project supportec by the Natural Science Foundation of Anhui Province,No 9741006 and Natural Science Foundation of Anhui Educational Commission,No.JL-97-077
文摘Aim The 3’-base specific polymerase chain reaction (3’- BS- PCR) method was es-tablished to investigate the relationship between the mutation of precore region of Hepatitis B virus(HBV) and the liver damage to the patients caused by HBV and the possibility of HBV precore gene in-tegration in liver cells。 Mdthods According to the DNA sequence of precore region of HBV,themethod of 3’- BS- PCR is applied to analyze the point mutation site 1896 of HBV precore in 126 clini-cal serum specimens and 23 hepatoeellular carcinoma (HCC) patients’ tissues and serum whose trmorshave been surgically excised and pathologically diagnosed.Rdsults The point mutation in site 1896 ofHBV precore has been successfully rates of preore gene of HBV in the 23 patients’ tissues and serum are52.2 % (12/23) and 30.4 % (7/23) respectively.Conclusion The established method for HBV ore-core mutation analysis is simple and results can well repeated.It has provided a new approach to clinicalHBV research and its relationship to liver damage.The results obtained suggested that HBV precoremutation exists in a wide range among serum and tissue of the patients infected by HBV and HCC pa-tients,and the pre-c gene of HBV can not be detected in the serum of 21.8% of the HCC patients(tissue HBV precore gene positive).We may deduce that there may be the integration of HBV precoregenee in the genome of liver cells,which may play an important role in the carcinogenesis of HCC.