Helicobacter Pylori Infection In Patients With Chronic Hepatitis C And Identification Of Helicobacter Hepaticus 16SrRNA In Different Tissues Of Digestive System From Various Strains Mice

Part 1 The seroprevalence of H.pylori in patients with chronic hepatitis C and the association with the virus loads and genotypes of HCV RNAAims: To investigate Helicobacter pylori infection in patients with the chronic hepatitis C and the synergy effect with HCV in progress of the the chronic hepatitis C. Methods: In this case-control study, cases were 376 patients with the chronic hepatitis C (252 patients with the chronic hepatitis C, 68 patients with chronic HCV-related cirrhosis and 56 patients with hepatocellular carcinoma). Controls were 327 healthy blood donors and 328 patients with chronic gastritis at the same period. All subjects were tested serum anti-H.pylori-IgG, anti- H.pylori-CagA-IgG and anti-H.pylori-VacA-IgG to determined whether there was H.pylori infection. Cases were tested the quantitation and genotype of HCV RNA. The serum anti-H.pylori-IgG was detected by ELISA and anti-H.pylori-CagA-IgG and anti-H.pylori-VacA-IgG were detected by Western-blot method. The serum quantitation of HCV RNA was detected by FQ-PCR and the genotype of HCV RNA was detected by PCR- micro-plate hybridization -ELISA. Results: H.pylori infection was more prevalent in patients with Chronic Hepatitis C (55.6%), chronic HCV-related cirrhosis (76.5%) and Hepatocellular carcinoma (78.6%) than that in healthy controls (43.4%) (p <0.01), and was similar to that in patients with the chronic gastritis (57.9%) (p> 0.05). Moreover, the seroprevalence of H.pylori in patients with chronic HCV-related cirrhosis and hepatocellular carcinoma was higher than that in patients with the chronic hepatitis (p<0.05). Typeâ… H.pylori infection in patients with chronic hepatitis C was 58.1%,and was significantly higher than that in healthy controls (22.5%) (p<0.01 ). Among the patients with chronic hepatitis C, typeâ… H.pylori infection in patients with chronic cirrhosis (76.9%) and hepatocellular carcinoma (79.5%) was more higher than that in chronic hepatitis (44.3%) (p <0.05). H.pylori infection aggravated with the progress of chronic hepatitis C. H.pylori infection in patients with different virus load of HCV RNA (+) was higher than that in healthy controls (p <0.01), but in patients with chronic hepatitis C were significantly but no significant difference between different virus loads (p> 0.05). H.pylori infection was 63.0%, 64.7%, 61.5% ,61.0% and 64.3% in patients with genotypes 1a, 1b, 2a, 2b and mixed/undeceted of HCV RNA, respectively. However,there was no significant difference in these genotypes (p> 0.05 ). Conclusion: H.pylori infection in patients with the chronic Hepatitis C was higher significantly and increased with the progress of the disease. Typeâ… H.pylori was the main strain in patients with the chronic hepatitis C. However, there was no significantly difference among patients of different virus loads and among patiens of differet genotypes. Part 2 Identification of H.pylori cagA, vacA and glmM genes and helicobacter species 16SrRNA in liver samples of patients with chronic hepatitis CAmis: To investigate the the role of H.pylori in the development of chronic hepatitis C. Methods: Serum anti-H.pylori-IgG was tested by ELISA in 34 patients with chronic hepatitis C. H.pylori-related genes(cagA, vacA and glmM) were detected by PCR with H.pylori associated genes-specific primers in liver samples of chronic hepatitis C patients with serum anti-H.pylori-IgG positive. Helicobacter genus-special 16SrRNA gene was detected by PCR with helicobacter genus-special 16SrRNA primers in liver samples of chronic hepatitis C patients with serum anti-H.pylori-IgG negative. Then, the amplified products of helicobacter genus-special 16SrRNA gene positive were identified by sequencing. The liver samples of Helicobacter genus-special 16SrRNA gene or helicobacter genus-special 16SrRNA gene was positive were isolated and cultured of bacteria. Results: Among 34 patients with chronic hepatitis C patients, serum anti-H.pylori-IgG were positive in 67.6% (23/34) and the seroprevalence of H.pylori was higher in patients with cirrhosis (10/14, 71.4%) and hepatocellular carcinoma (5 / 6, 83.3%) than that in chronic hepatitis (8 / 14, 57.1%) (p <0.05). H.pylori associated genes were found in 7 of 23 (30.4%) liver samples of chronic hepatitis C patients with serum anti-H.pylori-IgG positive. The positive rate of H.pylori-related genes in patients with hepatocellular carcinoma(4 / 5, 80.0%) was higher than that in that in patients with chronic hepatitis (1 / 8, 12.5%) and cirrhosis (2 / 10, 20.0%)( p <0.05), and the glmM gene was the main gene. Therefore, Helicobacter genus-special 16SrRNA gene was found in 1 of 11 liver samples of chronic hepatitis C patients with serum anti-H.pylori-IgG negative. Then, the sample amplified products of helicobacter genus-special 16SrRNA gene positive were sequenced and were found that the sample was similar to H.hepaticus with 92.0%. Conclusion: H.pylori-related genes were existed in liver samples of chronic hepatitis C in patients with H.pylori infection, and the positive rate of H.pylori-related genes in patients with hepatocellular carcinoma was higher than that in that in patients with chronic hepatitis and cirrhosis. Other helicobacter 16SrRNA genes were also existed in liver samples of patients with chronic hepatitis C. H.pylori and other helicobacter infection might play an important synergic role with HCV in the development of HCC.Part 3 Identification of H.hepaticus 16SrRNA in different tissues of digestive system from various strains miceAmis: To detect H. hepaticus special 16SrRNA gene in different tissues of digestive system from various strains mice infected by H. hepaticus in order to investigate the role of H. hepaticus on chronic hepatitis, hepatocellular carcinoma and typhlocolitis, as well as the lower-bowel carcinoma in mice. Methods: H. hepaticus infection was determined by serum antibodies against H. hepaticus, H. hepaticus antigen in stool and H. hepaticus specific 16SrRNA gene in stool in 102 different strains mice from 4 commercial animal center and 11 academic institutions of China. Then, H. hepaticus specific 16SrRNA gene was detected and the isolation of bacteria was performed in different tissues of digestive system from different strains mice infected. Results: H.hepaticus infection was found in 12 of 102 different strains mice, and the infection rate was 66.7% in A/J mice, significantly higher than that in C57BL/6 mice(5.1% ) and in BABL/C mice(7.0%) (p <0.01), but no significantly different to SCID mice rate (21.4%). Based on the results, H. hepaticus specific 16SrRNA gene was amplified in different tissues of digestive system from 12 mice infected H. hepaticus. The results showed that positive foundings of H. hepaticus specific 16SrRNA gene in different tissues of digestive system from the 12 mice was different. The positive of the cecal samples (66.7%) was significantly higher than that of liver samples (16.7% )(p<0.05) and that of gastric samples (8.3%)(p<0.01), and that of small intestine samples was 33.3%. Only 1 A/J mouse was found the suspicious colony in cecal sample by the isolation of bacteria from different tissues of digestive system confirmed that H. hepaticus specific 16SrRNA gene was positive. The suspicious colony was identificaed as H. hepaticus by the morphology, biochemical features, H. hepaticus specific 16SrRNA gene, as well as electron microscopy. Conclusion: H. hepaticus infection was found in different strains of mice (about 11.8%), and the infection rate of A/J mice was the highest (66.7%). H. hepaticus was colonized in cecal of mice. The isolation and culture to a suspicious strains identified confirmed H. hepaticus. The suspicious colony by isolation of bacteria from different tissues of digestive system was identificaed as H. hepaticus.