摘要
Golgi protein 73 (GP73) is a transmembrane protein on the Golgi apparatus and can be cut and released into the blood. In recent years, an increasing number of clinical studies have shown that the elevated serum GP73 level is closely related to liver diseases. And thus GP73 is expected to be used as a new serum marker for assessing progress of chronic liver diseases. Herein, the clinical application of serum GP73 in chronic hepatitis, liver fibrosis, liver cirrhosis and hepatocellular carcinoma with different etiologies was reviewed based on available literatures; and a research outlook in this field is made.
Subject(s)
Humans , Biomarkers , Carcinoma, Hepatocellular , Golgi Apparatus , Liver Cirrhosis , Liver Neoplasms摘要
We aimed to explore the associations between the age at which children undergo surgery for hypospadias and a range of social and clinical factors in a single center. Our aim was to promote the early surgical treatment of children with hypospadias. For a 6-year period, social and clinical data were collected from all children undergoing surgery to repair hypospadias in Children's Hospital of Chongqing Medical University (Chongqing, China), located in southwest of China. We analyzed the correlations between age at surgery and a range of social and clinical factors. A total of 1611 eligible cases were recruited, with a mean age of 54.3 months and a median age of 42 months: 234 cases (14.5%) were classified into a "timely operation" group, 419 (26.0%) cases into a "subtimely operation" group, and 958 (59.5%) cases into a "delayed operation" group. According to multivariate regression analyses, the higher the regional economic level, the closer the urethral opening to the perineum, and the higher the educational level of the guardians was, the younger the children were when they underwent the initial surgery for hypospadias; this was also the case for families without other children. Our subgroup analysis showed that the primary educational level of the guardians was a risk factor for subtimely surgery in their children (odds ratio [OR] = 1.52, 95% confidence interval [CI]: 1.08-2.15, P < 0.05). A lower regional economic level (OR = 1.87, 95% CI: 1.26-2.78, P < 0.01), a lower educational level of the guardians (OR = 3.84, 95% CI: 2.31-6.41, P < 0.01), and an anterior-segment urethral opening (OR
摘要
Background@#Our previous studies have shown that regulatory factor X5 (RFX5), a classical transcription regulator of MHCII genes, was obviously overexpressed in hepatocellular carcinoma (HCC) tumors. However, the role of RFX5 in the carcinogenesis and progress of HCC remains unknown. This study aimed to reveal its biological significance and the underlying mechanism in HCC.@*Methods@#RFX5 mRNA expression level and copy number variation in HCC tumors and cell lines were determined by analyzing deposited data sets in the Cancer Genome Atlas and Gene Expression Omnibus database. The biological significance of RFX5 in HCC was investigated by monitoring the colony formation and subcutaneous tumor growth capacity when RFX5 was silenced with lentiviral short hairpin RNA and CRISPR/Cas9 system in HCC cell lines. The downstream gene transcriptionally activated by RFX5 in HCC cells was determined by chromatin immunoprecipitation and luciferase reporter assay. The involvement of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (YWHAQ) in HCC development was further determined by performing colony formation rescue assay and subcutaneous tumor growth rescue experiment. The association of YWHAQ with recurrence-free survival of patients with HCC was assessed by Kaplan-Meier analysis. Moreover, apoptosis level and the protein level of p53 pathway were determined to reveal the mechanism of RFX5 in driving HCC development.@*Results@#RFX5 was amplified and highly overexpressed in HCC tumor tissues compared with the corresponding non-tumor tissues. The mRNA expression level of RFX5 was significantly correlated with its DNA copy number (r = 0.4, P < 0.001). Functional study demonstrated that RFX5 was required for both clonogenic forming in vitro and subcutaneous tumor growth in vivo of HCC cells. Further study identified YWHAQ, namely 14-3-3 tau, as a key downstream transcriptional target gene of RFX5, which was tightly regulated by RFX5 in HCC. Moreover, overexpression of YWHAQ largely rescued the clonogenic growth of HCC cells that was suppressed by RFX5 knockdown. In addition, overexpression of YWHAQ in primary tumor was linked to poor prognosis of patients with HCC. These results demonstrated that YWHAQ was a downstream effector of RFX5 in HCC. Notably, RFX5-YWHAQ pathway could protect cells from apoptosis by suppressing the p53 and Bax in HCC.@*Conclusion@#RFX5 is a putative HCC driver gene that plays an important role in the development and progression of HCC by transactivating YWHAQ and suppressing apoptosis.
摘要
BACKGROUND@#Our previous studies have shown that regulatory factor X5 (RFX5), a classical transcription regulator of MHCII genes, was obviously overexpressed in hepatocellular carcinoma (HCC) tumors. However, the role of RFX5 in the carcinogenesis and progress of HCC remains unknown. This study aimed to reveal its biological significance and the underlying mechanism in HCC.@*METHODS@#RFX5 mRNA expression level and copy number variation in HCC tumors and cell lines were determined by analyzing deposited data sets in the Cancer Genome Atlas and Gene Expression Omnibus database. The biological significance of RFX5 in HCC was investigated by monitoring the colony formation and subcutaneous tumor growth capacity when RFX5 was silenced with lentiviral short hairpin RNA and CRISPR/Cas9 system in HCC cell lines. The downstream gene transcriptionally activated by RFX5 in HCC cells was determined by chromatin immunoprecipitation and luciferase reporter assay. The involvement of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (YWHAQ) in HCC development was further determined by performing colony formation rescue assay and subcutaneous tumor growth rescue experiment. The association of YWHAQ with recurrence-free survival of patients with HCC was assessed by Kaplan-Meier analysis. Moreover, apoptosis level and the protein level of p53 pathway were determined to reveal the mechanism of RFX5 in driving HCC development.@*RESULTS@#RFX5 was amplified and highly overexpressed in HCC tumor tissues compared with the corresponding non-tumor tissues. The mRNA expression level of RFX5 was significantly correlated with its DNA copy number (r = 0.4, P < 0.001). Functional study demonstrated that RFX5 was required for both clonogenic forming in vitro and subcutaneous tumor growth in vivo of HCC cells. Further study identified YWHAQ, namely 14-3-3 tau, as a key downstream transcriptional target gene of RFX5, which was tightly regulated by RFX5 in HCC. Moreover, overexpression of YWHAQ largely rescued the clonogenic growth of HCC cells that was suppressed by RFX5 knockdown. In addition, overexpression of YWHAQ in primary tumor was linked to poor prognosis of patients with HCC. These results demonstrated that YWHAQ was a downstream effector of RFX5 in HCC. Notably, RFX5-YWHAQ pathway could protect cells from apoptosis by suppressing the p53 and Bax in HCC.@*CONCLUSION@#RFX5 is a putative HCC driver gene that plays an important role in the development and progression of HCC by transactivating YWHAQ and suppressing apoptosis.
摘要
Background@#DNA replication and sister chromatid cohesion 1 (DSCC1) (also called DCC1) is a component of an alternative replication factor C complex that loads proliferating cell nuclear antigen onto DNA during S phase of the cell cycle. It is located at 8q24 and frequently amplified in hepatocellular carcinoma (HCC). However, the role of DSCC1 in the carcinogenesis and progress of HCC has not been fully investigated. Here, we aimed to assert the importance of DSCC1 in the HCC.@*Methods@#In this study, copy number variation data and RNA sequencing data were used to calculate the DNA copy number and mRNA expression of DSCC1 in HCC. Quantitative polymerase chain reaction, Western blotting, and immunohistochemistry analysis were used to determine the mRNA and protein level of DSCC1 in HCC. The Kaplan-Meier analysis and univariate and multivariate Cox regression analysis were used to assess the association of DSCC1 with the overall survival (OS) of HCC patients. Moreover, lentiviral shRNA was used to knockdown DSCC1, and then, colony-forming assay, cell cycle assay, and cell proliferation assay were performed to evaluate the impact of DSCC1 silencing on HCC cell lines.@*Results@#We found that DSCC1 was amplified and highly expressed in HCC tumor tissues than in nontumor tissues. We then found that the overexpression of both mRNA and protein of DSCC1 was linked to the bad prognosis of HCC patients. Astonishingly, the protein level of DSCC1 was an independent prognostic factor for OS (hazard ratio, 1.79; 95% confidence interval, 1.17-2.74; P = 0.007). Furthermore, the clonogenic capacity of DSCC1-amplified HCC cell lines (MHCC-97H, MHCC-97L, and Hep3B) was significantly inhibited by transduction of a lentiviral shRNA that targets DSCC1. We also showed that knockdown of DSCC1 induced G0-G1 cell cycle arrest (increased from 60% to more than 80%) and greatly inhibited the proliferation of HCC cell lines.@*Conclusion@#These results suggest that DSCC1 is a putative HCC driver gene that promotes proliferation and is associated with poor prognosis in HCC.
Subject(s)
Female , Humans , Male , Middle Aged , Blotting, Western , Carcinoma, Hepatocellular , Genetics , Pathology , Cell Cycle , Genetics , Physiology , Cell Cycle Checkpoints , Genetics , Physiology , Cell Line, Tumor , Cell Proliferation , Genetics , Physiology , DNA Replication , Genetics , Physiology , Hep G2 Cells , Immunohistochemistry , Liver Neoplasms , Genetics , Pathology , Multivariate Analysis , Proportional Hazards Models , Real-Time Polymerase Chain Reaction摘要
<p><b>OBJECTIVE</b>To explore the correlation between single nucleotide polymorphisms (SNPs) of interleukin-28B (IL-28B) gene and the susceptibility to primary hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>A total of 300 histologically confirmed HCC cases (from November 2001 to April 2010) and 310 healthy controls with no history of chronic hepatitis B or hepatocellular carcinoma (2009-2010) were selected from a hospital in Guilin and a hospital in Beijing for this case-control study.139 HCC patients in the case group had complete clinical tracking data. All the subjects were Han Chinese, with no age or gender restrictions.2 ml peripheral blood samples were drawn from each subject with informed consent. SNP of rs12972991, rs4803223, rs8099917 and rs12979860 four loci in IL-28B gene were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF).</p><p><b>RESULTS</b>The frequencies of C allele at rs12972991, G allele at rs8099917 and G allele at rs4803223 were 6.7% (40/598), 7.9% (47/598) and 10.0% (59/588) respectively in case group; all higher than the corresponding frequencies in control group, separately 2.9% (18/618), 4.1% (25/616) and 3.6% (21/608). The differences were statistically significant (χ2=9.542, 7.858, 20.736, P values all<0.05). The above alleles could increase the risk of HCC, and the OR (95%CI) values were separately 1.67 (1.13-2.46), 1.49 (1.08-2.06) and 2.91 (1.79-4.72). The genotype frequencies of AC+CC at rs12972991, GT+GG at rs8099917, GA+GG at rs4803223 were 13.0% (39/299), 14.7% (44/299) and 19.0% (56/296) respectively in case group; while the frequencies were lower in control group, separately 5.8% (18/309), 8.1% (25/308) and 6.6% (20/304). The differences were statistically significant (χ2=9.319, 6.557, 20.948, P values all<0.05). These genotypes may increase the risk of HCC, and the adjusted OR (95%CI) values were 2.24 (1.31-3.83), 1.81 (1.14-2.88) and 2.90 (1.78-4.70), respectively. The stratified analysis of the clinical data indicated that the frequency of genotype GA+GG at rs4803223 was 50.0% (13/26) in patients of tumor thrombosis in portal vein (TTPV), higher than the frequency of genotype AA (21.1%, 23/109). The difference was statistically significant (χ2=8.965, P=0.003).</p><p><b>CONCLUSION</b>The results suggested that IL-28B gene polymorphisms was correlated to the susceptibility to HCC in Chinese Han ethnic population. Among them, GA + GG genotype at rs4803223 could increase the risk of TTPV in HCC patients.</p>
Subject(s)
Female , Humans , Male , Middle Aged , Alleles , Carcinoma, Hepatocellular , Genetics , Case-Control Studies , Genetic Predisposition to Disease , Genotype , Interleukins , Genetics , Liver Neoplasms , Genetics , Polymorphism, Single Nucleotide摘要
<p><b>OBJECTIVES</b>(1) To evaluate the prevalence, phenotypes and suppressive function of CD4+CD25+ regulatory T cells (Tregs) among the in peripheral blood mononuclear cells (PBMCs) and tumor-infiltration lymphocytes (TILs) from hepatocellular carcinoma (HCC) patients and patients with chronic hepatitis B. (2) To investigate the correlation between the frequency of CD4+CD25+ Tregs and clinical characteristics of HCC patients.</p><p><b>METHODS</b>PBMCs and TILs in 18 HCC patients, 10 chronic hepatitis B (CHB) patients and 15 healthy donors were evaluated for the phenotypes of CD4+CD25+ Tregs and the proportion of CD4+CD25+ Tregs as a percentage of the total CD4+ cells, by flow cytometric analysis with three or four color staining. The relationship between the frequency of CD4+CD25+ Tregs and tumor TNM stages was analyzed. The CD4+CD25+ Tregs and CD4+CD25- T cells were isolated from PBMC of HCC patients and donors. The suppressive function of CD4+CD25+ Tregs was analyzed.</p><p><b>RESULTS</b>The percentages of CD4+CD25+ Tregs of the HCC patients (6.38% +/- 6.30%) and CHB patients (4.29% +/- 1.82%) were significantly higher than those of the healthy donors (1.58% +/- 0.55%, P less than 0.01). Among the TILs, the percentage of CD4+CD25+ Tregs was higher (t = 4.39, P < 0.01). There were significant differences in the prevalence of CD4+CD25+ Tregs in early and advanced stage HCCs (stage II vs. III, P less than 0.05; stage II vs. IV P < 0.01). The proliferative capacity of CD4+CD25- T cells was inhibited by the presence of CD4+CD25+ T cells in a dose-dependent manner where the level of suppression was correlated to the ratio of the two-cell populations.</p><p><b>CONCLUSION</b>These results suggest that the increase in frequency of CD4+CD25+ Tregs might play a role in the suppression of the immune response against HCC, which may contribute to the HCC cells that escaped from immunological surveillance.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Carcinoma, Hepatocellular , Metabolism , Interleukin-2 Receptor alpha Subunit , Liver Neoplasms , Metabolism , T-Lymphocytes, Regulatory , Allergy and Immunology摘要
<p><b>OBJECTIVE</b>To gain some insights into the critical events relating to HBV transcriptional regulation by comparing HBV replicative characteristics in different cell lines.</p><p><b>METHODS</b>Hepatic cell lines QSG-7701 and HepG2 were transfected with plasmid PUC18-HBV 1.2 by standard calcium phosphate precipitation method, and 1.0 microg pSEAP2-control vector was included in the transfection procedures to serve as an internal control monitoring the transfection efficiency. Hepatitis B surface antigen (HBsAg) in the medium was detected by ELISA method and HBV DNA was quantitated using fluorescent quantitative PCR. The intracellular HBsAg and HBcAg were detected with immunofluorescent staining. The gene expression profiles of QSG-7701 and HepG2 were compared using oligonucleotide microarray; partial differentially expressed genes were verified with quantitative RT-PCR.</p><p><b>RESULTS</b>In the medium of the cultured HepG2 cells, HBsAg and HBV DNA could be detected 6 days after the transfection, whereas in QSG-7701 cells, the HBsAg and HBV DNA could be detected for 2 weeks. The HBV DNA in the culture medium of QSG-7701 was about 50 times more than that of the HepG2 cells which were kept in 1 x 10(7)copies/ml(-3) x 10(7)copies/ml for 0 to 10 days after the transfection. On the 4th day after the transfection, 20% to 30% of the QSG-7701 cells were positive with HBsAg and HBcAg immunofluorescent staining. The gene microarray analysis showed that most transcription factors involved with HBV life cycle in QSG-7701 and HepG2 cells had similar levels, whereas some factors involved with HBV transcriptional regulation and core particle disassembly, such as interleukin-6 (R=5.1340), retinoid X receptor, alpha (R=5.1268), hepatic leukemia factor (R=3.2538), serine protease PRRS23 (R=2.8356), hepatitis B virus x interacting protein (R=0.4939), serine protease inhibitor Kazal type 1 (R=0.0740) and matrix metalloproteinase 3 (negative in QSG-7701) were all differentially expressed by HepG2 cells.</p><p><b>CONCLUSION</b>Different than HepG2 cells, the QSG-7701 cells could support a high level and relatively stable HBV replication after HBV DNA transient transfection. The HBV core particles were probably recycled in the QSG-7701 cells. The differential gene expressions between QSG-7701 and HepG2 might explain the mechanism of the different HBV replication patterns. Hepatic cell line QSG-7701 might serve as a useful tool for HBV transcriptional regulation research.</p>
Subject(s)
Humans , Cell Line , Gene Expression Regulation, Viral , Genetic Vectors , Genome, Viral , Hep G2 Cells , Hepatitis B virus , Genetics , Physiology , Virus Replication摘要
<p><b>OBJECTIVES</b>To investigate the correlation between MAGE-A1 mRNA expression and genic demethylation in hepatoma cell lines.</p><p><b>METHODS</b>Total RNA and genomic DNA were prepared from 10 human hepatoma cell lines. MAGE-1 mRNA expression was determined with RT-PCR and the level of genome-wide demethylation was evaluated by enzyme digestion and Southern blot assay. The genomic DNA was digested by HpaII, then the promoter of MAGE-A1 gene was amplified with primers CDS21, EDP4 and CDS20. EDP4 and the PCR products were further hybridized with a probe to detect the methylation in the promoter of the MAGE-A1 gene. HLA-A locus was typed using SSP kit.</p><p><b>RESULTS</b>In cell lines QGY-7703, SMMC-7721, HLE, BEL-7402, BEL-7404 and BEL-7405, MAGE-A1 mRNA expression was positive and cell differentiation was moderate or low. In cell lines of HepG2 2.2.15, HepG2, QGY-7701 and Huh7, MAGE-A1 mRNA expression was negative and cell differentiation was well or moderate. The level of genomic demethylation in MAGE-A1 mRNA positive cell lines was much higher than that in MAGE-A1 mRNA negative cell lines (t = 2.896, P = 0.02). The methylation analysis showed that methylation in the promoters of MAGE-A1 gene of HepG2 2.2.15, HepG2, QGY-7701 and Huh7 was high, and that methylation in those of SMMC-7721, HLE, BEL-7402, BEL-7404, and BEL-7405 was low.</p><p><b>CONCLUSION</b>The results suggest that MAGE-A1 mRNA expression in the human hepatoma cell lines is associated with genic hypomethylation.</p>
Subject(s)
Humans , Antigens, Neoplasm , Carcinoma, Hepatocellular , Genetics , Allergy and Immunology , Metabolism , DNA Methylation , Liver Neoplasms , Genetics , Allergy and Immunology , Metabolism , Melanoma-Specific Antigens , Neoplasm Proteins , Genetics , RNA, Messenger , Genetics , Tumor Cells, Cultured摘要
<p><b>OBJECTIVES</b>To investigate the expression of melanoma-associated antigen 1 (MAGE-1) in Chinese hepatocellular carcinoma (HCC) patients and to determine the existence and distribution of single nucleotide polymorphisms (SNP) of MAGE-1 gene.</p><p><b>METHODS</b>Total RNA was extracted from cancer tissues and adjacent tissues from 19 HCC patients and the expression of MAGE-1 mRNA was examined by using RT-PCR. The PCR products were sequenced to analysis the gene variation. Genomic DNA was extracted from cancer tissues, adjacent tissues and peripheral blood cells of 19 HCC patients, as well as from peripheral blood cells of 23 healthy donors. The PCR product of MAGE-1 DNA was sequenced to determine the existence and distribution of SNPs of MAGE-1 gene.</p><p><b>RESULTS</b>9 of 19 (47.4%) tumor tissues and none of adjacent tissue from HCC patients expressed MAGE-1 mRNA. There were three kinds of gene variations of cDNA in 9 MAGE-1 mRNA positive patients. One type was named type I including 1 patient, which sequence is as same as that of the GenBank M77481. The other was named TGA type including 5 patients, which involved three nucleotide changes (C159T, A272G and G393A) and result in two amino acid changes (T32A and R72Q). Another one was named GTG type including 3 patients, which involved three nucleotide changes (A272G, C991T, A1125G) and result in only one amino acid changes (T32A). According to the analysis of genomic DNA, above three types were not specific mutations of tumor tissue, but were SNPs. These SNPs types were distributed in HCC patients and normal donors with the frequencies of 26.3% (5/19) and 60.9% (14/23) for type I, 57.9% (11/19) and 47.8% (11/23) for TGA type, and 21.1% (4/19) and 21.7% (5/23) for GTG type, respectively. The sequences of two new SNPs had been deposited in GenBank with the accession numbers of AF463515 and AY148486. In male population, the distributions of SNPs were not correlated to HCC suffering or MAGE-1 expression. Several new HLA-restricted epitopes were probably resulted from SNPs existing. The three-dimensional models of MAGE-1 proteins of type I and TGA type was established by using computer software.</p><p><b>CONCLUSION</b>The expression rate of MAGE-1 gene in Chinese HCC patients is high. Three SNP types of MAGE-1 gene exist in Chinese population. The three-dimensional models of MAGE-1 proteins were obtained by computer processing. These results will be helpful to developing MAGE-1 peptide-vaccine for HCC immunotherapy.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Amino Acid Sequence , Antigens, Neoplasm , Carcinoma, Hepatocellular , Genetics , Liver Neoplasms , Genetics , Melanoma-Specific Antigens , Molecular Sequence Data , Neoplasm Proteins , Genetics , Polymorphism, Single Nucleotide , RNA, Messenger , T-Lymphocytes, Cytotoxic , Allergy and Immunology摘要
<p><b>OBJECTIVE</b>To investigate the correlation between impaired non-viral specific immune function of dendritic cell (DC) and viral clearance and cytotoxic T lymphocyte (CTL) response to HBV or HCV in patients with HBV and HCV coinfection.</p><p><b>METHODS</b>Twenty-five patients with HBV and HCV coinfection were investigated in this study. In 1994 and 2002, biochemical and virological markers and quantitative serum HBV DNA and HCV RNA levels were detected in these patients. According to the virus clearance status, these patients were divided into 4 groups: 14 patients with both HBV and HCV clearance (Group A), 6 patients with HCV clearance only (Group B), 3 patients with HBV clearance only (Group C), and 2 patients with persistent infection of HBV and HCV (Group D). Phenotypes and immune functions of monocyte-derived DCs were compared between these groups. 51Cr release assay were used to measure CTL response to epitopes derived from HBV, HCV or influenza virus (as positive control) in HLA-A2+ patients.</p><p><b>RESULTS</b>Impaired non-viral specific immune functions of DCs were observed in group B, C and D compared with group A and normal donors (Group N). These impaired functions included CD86 decreasing expression and lower capacity to stimulating allogenic T cells and uptaking antigen. The specific CTL response to HBV- and HCV-derived peptides could be induced in group A (12/12). The specific CTL response to HBV-derived peptides or to HCV-derived peptides could be induced in group C (3/3) or B (5/5), respectively. But the specific CTL response to both of two HBV-derived peptides or two HCV-derived peptides could not be induced in group C (0/3) or B (0/5), respectively. And no CTL response to HBV or HCV-derived peptides could be induced in groups D (0/1) and N (0/4).</p><p><b>CONCLUSION</b>1. The results suggest that specific CTL response to HBV or HCV play a vital role in the viral clearance. 2. The DCs with impaired non-viral specific immune functions exist in chronic patients with HBV and/or HCV infection, but do not interfere with clearance and CTL response to HBV or HCV. It is reasonable to speculate that impaired functions of DCs result from viral infection.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Dendritic Cells , Allergy and Immunology , Hepacivirus , Allergy and Immunology , Hepatitis B virus , Allergy and Immunology , Immunophenotyping , Lymphocyte Culture Test, Mixed , T-Lymphocytes, Cytotoxic , Allergy and Immunology摘要
<p><b>OBJECTIVE</b>To construct a series of recombinant replication competent plasmids of hepatitis B virus (HBV) full-length genome with varying mutations of precore and basic core promoter.</p><p><b>METHODS</b>The plasmid containing the entire HBV (1.2 copies) genome was used to construct objective plasmids. The objective competent vectors were constructed by molecular cloning and PCR-based site-directed mutagenesis in vitro and confirmed with sequence analysis. After introducing the plasmids into hepatocellular carcinoma cell line (Huh7) by calcium phosphate transfection, the culture supernatant was collected to detect HBsAg and HBV DNA to analyze viral replication and expression.</p><p><b>RESULTS</b>Ten competent vectors with varying precore and basic core promoter mutations were constructed. After transfecting into Huh7, the vectors replicated and secreted viral particles.</p><p><b>CONCLUSIONS</b>Ten full-length competent HBV recombinants were obtained, which were harbouring varying mutations within precore and basic core promoter.</p>
Subject(s)
Humans , Carcinoma, Hepatocellular , Metabolism , Pathology , Cell Line, Tumor , Genetic Vectors , Genome, Viral , Hepatitis B Core Antigens , Genetics , Hepatitis B virus , Genetics , Physiology , Liver Neoplasms , Metabolism , Pathology , Mutagenesis, Site-Directed , Plasmids , Recombinant Proteins , Genetics , Metabolism , Transfection , Viral Core Proteins , Genetics , Virus Replication , Genetics摘要
<p><b>OBJECTIVE</b>To understand HBV serotypes and genotypes epidemiology in a northern city and a southern city in China.</p><p><b>METHODS</b>Using polymerase chain reaction (PCR) and direct sequencing of HBV DNA PCR products, the serotypes and genotypes of HBV in 530 from HBsAg positive samples. The enrolled patients were from Harbin, a northern city and Lianjiang, a southern city in China.</p><p><b>RESULTS</b>Comparison of the serotypes and genotypes of HBV between Harbin and Lianjiang showed that adrq+ was the most predominant hepatitis B virus serotype in both Harbin and Lianjiang (87.2% and 73.5%,respectively), adw2 was the next (12.0% and 25.7%, respectively); genotype C was the most frequent in Harbin and Lianjiang (87.8% and 73.2%, respectively), and genotype B was the next (12.2% and 26.1%, respectively) only 1 patient was infected by genotype D, and 1 patient was found to be co-infected by genotype B and C in Lianjiang.</p><p><b>CONCLUSION</b>The results suggest that the percentage of HBV serotypes and genotypes between Harbin and Lianjiang was significantly different (P less than 0.001), but the main HBV serotype and genotype of the two cities were similar.</p>
Subject(s)
Humans , China , DNA, Viral , Genetics , Genotype , Hepatitis B Surface Antigens , Blood , Hepatitis B virus , Classification , Genetics , Polymerase Chain Reaction , Serotyping摘要
<p><b>BACKGROUND</b>To study the relationship between HCV infection and the development of type II diabetes mellitus.</p><p><b>METHODS</b>1. The case record files of 126 patients with chronic hepatitis C vs. 227 with chronic hepatitis B were reviewed and the laboratory and demographic data were extracted. 2. Anti-HCV and HBsAg were determined for 160 type II diabetes patients and 223 healthy adults by ELISA.</p><p><b>RESULTS</b>1. The occurrence of diabetes in patients with chronic hepatitis C was 19.05%, higher than 8.37% in patients with chronic hepatitis B (P<0.01). Age and HCV infection were independent risk factors for diabetes. 2. Five patients with type II diabetes were anti-HCV positive (3.12%) while none of the 223 healthy adults was anti-HCV positive (P<0.05). Seven patients with diabetes (4.37%) and 12 healthy adults (5.38%)were HBsAg positive (P>0.05).</p><p><b>CONCLUSIONS</b>1. The occurrence of diabetes was significantly higher in patients with HCV related liver disease than in patients with HBV related liver disease. 2. The occurrence of anti HCV was higher in diabetes patients than in healthy adults. HCV may play a role in the development of diabetes mellitus.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , China , Epidemiology , Comorbidity , Diabetes Mellitus, Type 2 , Epidemiology , Virology , Hepatitis B, Chronic , Epidemiology , Hepatitis C, Chronic , Epidemiology , Prevalence , Random Allocation , Risk Assessment , Risk Factors摘要
<p><b>BACKGROUND</b>To investigate sequence of the complete E region of genotype 2a hepatitis C virus genome and to set up the basis for the further study on biological functions of envelope proteins of HCV.</p><p><b>METHODS</b>Two cDNA fragments of 770bp, 1,100bp long, from serum of a patients infected HCV, were amplified by reverse transcription nested polymerase chain reaction (RT-nPCR). After being digested with two restriction endonucleases, the two fragments were cloned into JM105 respectively, then sequenced.</p><p><b>RESULTS</b>The two corresponding nucleotide sequences were obtained and ligated. The complete nucleotide sequences of envelope region and corresponding deduced amino acid sequences were compared with those of HCV-1, HC-C2, HCV-BK, HC-J6 and HC-J8. The homology of nucleotide sequence of the isolated strains was 60.5%, 60.1%, 59.7%, 87.8% and 67.5% respectively; the amino acid sequence homology to the isolated strains was found to be 67?3%, 66.4%, 65.0%, 87.8%, 79.0%, respectively.</p><p><b>CONCLUSIONS</b>The results indicate that the isolated strain (HCV-JS) belongs to genotype 2a, but there is heterogeneity between HCV-JS and HC-J6 strain.</p>
Subject(s)
Humans , Amino Acid Sequence , Cloning, Molecular , DNA, Complementary , Genetics , DNA, Viral , Genetics , Genotype , Hepacivirus , Genetics , Hepatitis C , Virology , Molecular Sequence Data , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Viral Envelope Proteins , Genetics , Viral Nonstructural Proteins , Genetics摘要
<p><b>OBJECTIVE</b>To observe the differentiation of bone marrow stem cells in rat hepatic fibrogenesis environment into hepatocytes.</p><p><b>METHODS</b>Rat hepatic fibrosis was induced by subcutaneous injection of CCl4. Bone marrow stem cells with Thy positive, CD3 and CD45RA negative were enriched from the bone marrow by fluorescence-activated cell sorting. The bone marrow stem cells were labeled with PKH26-GL, and then autotransplanted. After six weeks, albumin, ck8 and a-smooth muscle actin expression were determined by immunocytochemistry.</p><p><b>RESULTS</b>The PKH26-GL labeled cells expressed albumin and ck8, but did not express a-smooth muscle actin in hepatic fibrogenesis environment.</p><p><b>CONCLUSION</b>Bone marrow stem cells in hepatic fibrogenesis environment can differentiate into hepatocytes, but can't differentiate into myofibroblasts</p>
Subject(s)
Animals , Male , Rats , Bone Marrow Cells , Pathology , Cell Differentiation , Physiology , Environment , Hematopoietic Stem Cells , Pathology , Hepatocytes , Pathology , Liver Cirrhosis , Pathology , Rats, Sprague-Dawley摘要
<p><b>OBJECTIVE</b>To study whether dendritic cells (DCs) derived from the peripheral blood in chronic hepatitis B patients can induce specific T cell immune response.</p><p><b>METHODS</b>(1)The subjects were divided into 3 groups: chronic hepatitis B group (CHB), acute hepatitis B group (AHB), and normal donor group (ND). The peripheral blood mononuclear cells (PBMCs) isolated from those subjects were stimulated with HBcAg 18 to 27 CTL epitope peptide, and intracellular cytokine staining (ICCS) was used for detecting IFN-gamma, IL-2 and TNF-alpha produced by CD8+ T cell. (2) DCs generated from PBMCs were pulsed with HBcAg 18 to 27 CTL epitope peptide, then were cocultured with autologous lymphocytes for 10 days to induce antigen-specific T cell, which was assessed by ICCS and cytotoxic assay.</p><p><b>RESULTS</b>(1) The memory effect of the PBMCs from AHB group to HBcAg 18 to 27 CTL epitope peptide was stronger than that from CHB or ND group (t=2.508-3.305, P<0.05). (2)After lymphocytes were cocultured with DC treated with HBcAg 18 to 27 CTL epitope peptide, antigen-specific T cell effect was induced. And the killing rates were (57.0+/-23.0)%, (49.5+/-20.2)%, (21.8+/-12.9)% at the effector/target of 30:1, 10:1, 3:1, which were higher than that in control group.</p><p><b>CONCLUSIONS</b>The memory T cells against HBV antigen lacks in CHB patients. DCs from CHB patients pulsed with HBcAg 18 to 27 epitope peptide can induce HBV antigen-specific T cell, which can kill specific target cells and produce cytokines involved in virus clearance.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , CD8-Positive T-Lymphocytes , Allergy and Immunology , Cells, Cultured , Dendritic Cells , Allergy and Immunology , Virology , Epitopes, T-Lymphocyte , Allergy and Immunology , Hepatitis B Core Antigens , Allergy and Immunology , Hepatitis B virus , Genetics , Allergy and Immunology , Hepatitis B, Chronic , Allergy and Immunology , Leukocytes, Mononuclear , Allergy and Immunology , T-Lymphocytes, Cytotoxic , Allergy and Immunology摘要
<p><b>OBJECTIVE</b>(1) To investigate the expression and gene diversity of the 7 major cancer/testis (CT) antigens, MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1, in hepatocellular carcinoma (HCC). (2) To analyze the correlations between the clinical characters and CT antigens' expression.</p><p><b>METHODS</b>The cancer and para-cancer tissues were collected from 30 HCC patients. The mRNAs of seven CT antigens were detected by reverse transcription-polymerase chain reaction (RT-PCR) with the specific primers. The PCR products were sequenced to analyze the CT genes.</p><p><b>RESULTS</b>The MAGE-1, MAGE-3, MAGE-4, MAGE-10, NY-ESO-1, SSX-2 and SCP-1 were expressed in 66.7%, 70.0%, 20.0%, 36.7%, 40.0%, 33.3% and 33.3% of the tumor tissues from HCC patients respectively, however, they were not expressed in the para-cancer tissues. Among the 30 patients investigated, 90.0% expressed one CT gene at least, 70.0% expressed two CT genes, and 53.3% expressed three CT genes of the seven CT genes. The coding genes of these CT antigens were highly conserved between in Chinese patients and patients abroad. There were discernible correlations between alpha-fetoprotein level and MAGE-10 or SCP-1 expression level, as well as between average age and MAGE-3 or SSX-2 expression levels (P<0.05).</p><p><b>CONCLUSIONS</b>With a highly conserved coding gene, seven CT antigens were expressed in 20.0% - 70.0% of Chinese HCC patients. CT antigens' expression had correlations with some clinical characters.</p>
Subject(s)
Female , Humans , Male , Antigens, Neoplasm , Genetics , Carcinoma, Hepatocellular , Genetics , Allergy and Immunology , Gene Expression Regulation, Neoplastic , Genetics , Liver Neoplasms , Genetics , Allergy and Immunology , Melanoma-Specific Antigens , Neoplasm Proteins , Genetics , RNA, Messenger , Genetics摘要
<p><b>OBJECTIVES</b>To investigate the profile of liver histological damage after autologous adoptive immunotherapy with cytokine-induced killer cells (CIK cells) in patients with chronic hepatitis B (CHB).</p><p><b>METHODS</b>16 CHB patients were randomly enrolled and received autologous adoptive immunotherapy, then followed up 52 weeks. Liver samples were taken from the patients to evaluate the degree of inflammation and fibrosis. The markers of hepatitis B virus and liver function were also detected.</p><p><b>RESULTS</b>4 patients had two liver-biopsied samples before therapy and after 52 weeks follow-up. One patient's histological assessment revealed a significant improvement in intralobular necroinflammation (G2 --> G1) and fibrosis (S2 --> S1). The others failed to show obvious changes in liver histology. After 10 days culture in vitro, phenotypic characterization of CIK cells changed significantly by flow cytometry. The percentage of CD4+ cells decreased gradually, while the percentage of CD8+ cells increased from 20% to 60% - 80%. After 52 weeks follow-up, HBV DNA was negative (HBV DNA<4pg/ml in serum) in 6 out of 14 patients. The rates of both HBeAg/anti-HBe seroconversion and alanine aminotransferase normalization were 42.86%(6/14). There was no HBsAg/anti-HBs seroconversion. There was few severe treatment-related adverse events.</p><p><b>CONCLUSION</b>Autologous adoptive immunotherapy doesn't induce the damage of liver histology in chronic hepatitis B patients, which inhibits hepatitis B virus replication by a certain noncytotoxic mechanism.</p>
Subject(s)
Adolescent , Adult , Aged , Humans , Middle Aged , CD4-CD8 Ratio , DNA, Viral , Hepatitis B, Chronic , Allergy and Immunology , Pathology , Therapeutics , Immunotherapy, Adoptive , Liver , Pathology摘要
<p><b>OBJECTIVE</b>To investigate the relationship between different genotypes of hepatitis B virus (HBV) and the severity of liver diseases.</p><p><b>METHODS</b>The S nucleotide sequences of HBV strains isolated from plasma samples of 284 patients were detected and compared. Among them, 87 patients were HBV asymptomatic carriers (ASC), 157 chronic hepatitis B (CHB), 22 liver cirrhosis (LC), and 18 hepatocellular carcinoma (HCC).</p><p><b>RESULTS</b>Genotypes B and C were predominant, with a 26.1% proportion and a 63.2% proportion respectively. The percentage of genotypes B and C in patients with ASC, CHB, LC, and HCC were significantly different (x(2)=15.09, P<0.001). Compared with genotype B, genotype C was more common in patients with CHB and HCC (59.6% vs 43.2%, chi(2)=10.87, P<0.001; 7.7% vs 1.4%, x(2)=7.41, P<0.001), but in patients with LC there was no different (7.7% vs 8.1%, chi(2)=1.29, P>0.05).</p><p><b>CONCLUSION</b>This study suggests that genotype B and C are predominant. And genotype C may induce more severe the liver inflammation than genotype B may do.</p>