Dissertation > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors

Influence of Hepatitis C Virus Genotypes and F Protein on Chronic Hepatitis C Virus Infection and Hepatocellular Carcinom Cell Apoptosis

Author YangJingJing
Tutor ZhangYun
School Nanjing Medical University
Course Epidemiology and Biostatistics,
Keywords Hepatitis C virus HCV genotype F protein antibody HCV persistent infection Self-limiting Clear Chronic Double shift F protein HepG2 Apoptosis Tumor necrosis factor α Flow cytometry DNA Ladder
CLC R735.7
Type Master's thesis
Year 2009
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Hepatitis C caused by the hepatitis C virus (HCV) infection was global distribution. According to WHO estimates, there are around 180 million HCV-infected patients, and the growing trend of HCV infection rate is much higher than the general population, especially in intravenous drug users (IDUs), is an important source of infection. As high as 75% to 85% rate of chronic hepatitis C due to HCV infection can lead to chronic liver inflammation, necrosis and fibrosis, some patients may develop cirrhosis and even hepatocellular carcinoma, constitutes a serious threat to human health and life . HCV chronic infection is the result of the interaction of virus mutation and host immune. HCV genome showed a higher variability of performance prevail dominant virus strain (quasi-species) variation and different regions different genotypes. Variation of the main purpose is to evade the host immune surveillance, which can persist in the host body, the formation of a chronic infection. HCV genotype distribution there are geographic differences, most of our major popular the HCV type of genotype 1b, followed for genotype 2a. Genotype 1b infection rate in the southern city of more than 90%. Domestic and international studies have shown that genotype 1b chronic hepatitis, cirrhosis and liver cancer in the vast majority, suggesting that genotype 1b HCV infection, chronic. The hepatitis C virus is a flavivirus, is about 9.6kb, divided into the 5 'untranslated region (UTR), an open reading frame (ORF) and the 3' untranslated region, coding for a single-stranded positive RNA a composite precursor protein by more than 3000 amino acids, is cleaved in the host signal peptidase and the role of the viral protease to generate at least 10 species of gene product: the four structural proteins (Core, E1, E2, p7), and six non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). Dozen proteins encoded by HCV, core protein (Core, referred to as C) is the HCV poly-protein precursor produced by cell signal peptidase cleavage of the non-glycosylated protein, composed of 191 amino acids (aa). The amino acid sequence of the protein is very conservative, and the regulation of multiple cellular genes, the ability to affect cell growth, proliferation and apoptosis. Foreign scholars in recent years, was found in the serum of patients infected with HCV, HCV F protein, is produced by another product of the gene encoding the HCV C, it is shifted by C codon nucleotide. In the highly conserved nature of the different genotypes of HCV, and in the presence of the antibody in vivo in patients, the F protein plays an important role in the HCV life cycle. 2003 Boulant studying the the HCV 1b C gene expression in E.coli found the the double shift F protein (ARFP / DF). DF AUG protein 1b C gene promoter, the C gene reading frame translated into 42 aa, 2/1 frame shift occurs, and then translate the C 1 reading frame to reach 144 aa happen again shift, re-translation, according to the C gene reading frame until the terminator. DF is part of the F gene and portion C of the chimeric gene product is an FC chimeric ribosome occurred twice shift in its generation process. At home and abroad there is no function of DF. F antibody and HCV infected chronic relationship as well as the relationship between HCV1b type DF protein with hepatocellular carcinoma (HCC) in order to study the distribution of HCV patients to explore HCV genotype HCV genotype F protein antibody drug in Jiangsu . We conducted the study as follows: the first part of the hepatitis C virus genotype F protein antibody Objective: To study the relationship between HCV genotype and F protein antibody among drug users in HCV infection, chronic hepatitis C virus infection among drug users . Methods: The subjects of this study the Nanjing compulsory treatment HCV antibody-positive Addicts 360 people, divided into 168 persistently infected group (HCV RNA positive blood test for HCV RNA and ALT levels, ALT ≥ 40 U / L), and self-limiting Clear group of 192 people (HCV RNA negative, ALT, lt; 40 U / L). HCV RNA genotyping assay kit HCV and enzyme-linked immunosorbent method (MUREX HCVSerotyping 1-6 Assay) to detect HCV genotype and HCV serotypes were applied; express the laboratory purified recombinant protein HCV-F/GST as antigen coated enzyme-linked reaction plate, two groups of serum samples to be tested for the primary antibody, horseradish peroxidase-conjugated anti-human IgG antibody as the secondary antibody as a background control GST protein hole, ELISA indirect method were detected in both groups serum. Results: Demographic characteristics by comparing HCV persistent infection and self-limiting cleanup between HCV genotyping and F protein antibody distribution differences come: persistent infection group and self-limiting clear group differences in gender composition between statistically significance; persistently infected with genotype 1 group and the mixed genotypes percentage than clear set of slightly higher self-limiting, genotype 2 opposite. Between the two groups genotyping constitute a clear set of slightly higher (36.3% vs 27.6%); significant difference (P lt; 0.05); the group persistently infected with HCV-F antibody positive rate than self-limiting univariate and multivariate Logistic regression analysis found that men (OR = 1.43; 95% CI, 1.03 - 2.49; P = 0.032) in patients infected with HCV genotype 1 (OR = 4.18; 95% CI ,2.14-8 .13; P = 0.0001), HCV -F antibody positive (OR = 1.73; 95% CI ,1.08-2 .47; P = 0.047) increase in chronic HCV infection risk. Conclusion: among drug users in male patients infected with HCV genotype 1 or HCV-F antibodies more likely to lead to chronic HCV infection. Useful for the clarification of the HCV susceptibility characteristics of the Chinese population and infection abroad there is no HCV-F antibody and HCV infection outcome reports, and drug crowd HCV genotype infection presents a complex trends, carry chronic HCV infection outcome Factors Influencing the outcome of the possible mechanism of vaccine development, individualized prevention and treatment, control the country of HCV infection has important theoretical and practical significance. The second part of the F protein of hepatitis C virus genotype 1b shift of human hepatocellular carcinoma cell line HepG2 apoptosis Objective: HCV genotype 1b chronic hepatitis, cirrhosis and liver cancer in the majority, while at home and abroad studies have reported no HCV 1b double shift F (DF) protein on apoptosis of hepatoma cells. This part of the main study of hepatitis C virus genotype 1b DF protein on apoptosis of hepatocellular carcinoma cell line HepG2. Method: The eukaryotic expression recombinant plasmid pcDNA3.0-DF-EGFP using Lipofectamine 2000 transfection of human hepatocellular carcinoma cell line HepG2 Let pcDNA3.0-C-EGFP-HepG2 positive control, pcDNA3.0-HepG2 as the negative control and non-transfected HepG2 blank control, Act-D, TNFα-induced apoptosis in the four cell lines, Annexin V-FITC/PI double staining rate of tumor cell apoptosis and apoptosis DNA Ladder detection HCV 1b DF protein further verify the effect of the biological functions of the liver cell apoptosis. Results: pcDNA3.0-DF-EGFP-HepG2 cell lines apoptosis faster than pcDNA3.0-C-EGFP-HepG2 but relatively negative control group and the non-transfected HepG2 cells was significantly delayed apoptosis The incidence was significantly lower than the negative control and blank control cell lines (P lt; 0.001). Conclusion: Since the introduction and expression of exogenous gene HCV 1b DF, inhibit apoptosis of HepG2 cells.

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