Establishment Of The Models Of Different Strains Of Mice Infected With Helicobacter Hepaticus And Analysis Of The Seroprevalance H.hepaticus-igG Antibody In Patients With Chronic Hepatitis B

Part1Establishment of the models ofdifferent strains of laboratory mice infected with Helicobacterhepaticus and analysis of pathological featuresObjective:Application of the different strains of mice infected withH.hepaticus Standard strain ATCC51450,to observe H.hepaticus colonizationand pathological features, to obtain stable animal models infected withH.hepaticus.Methods:SPF-class male BALB/cCr,SCID/Cr and C57BL/6Cr micewhich had confirmed no H.hepaticus infection were inoculated H.hepaticusstandard strain ATCC514500.2ml bacterial suspension(1×108CUF/ml),inoculated for3times with48hours intervals,the control groupwas fed with the same volume of PBS. Mice which lasted for12hours wereexecuted at1,3,6,9and12months since last after H.hepaticus inoculation(n=5).Serum were taken for H.hepaticus-IgG and mice esophagus,stomach, jejunum,ileum, cecum,colon,liver and pancreas tissue were taken for histopathologyexamiantion,Micro-aerobic bacteria isolated,cultured and identification andH.hepaticus specific16SrRNA gene amplification.Results: The seroprevalance H.hepaticus-IgG antibody in BALB/cCrmice were highest. The seroprevalance H.hepaticus-IgG antibody in BALB/cCrmice infected with H.hepaticus were all100%from1months to12months.Theseroprevalance H.hepaticus-IgG antibody in C57BL/6Cr mice were between 40~60%, The seroprevalance H.hepaticus-IgG antibody in SCID/Cr miceinfected with H.hepaticus from1months to12months were not detected.Antibody levels in BALB/cCr and C57BL/6Cr mice infected withH.hepaticus reached a peak at6months, then gradually decline. Thecolonization rates of H.hepaticus in cecum in BALB/c Cr and SCID/Cr miceinfected with H.hepaticus at1months were all80%, then continuedcolonization in the cecum, colonization rates in cecum from3to12monthswere all (100%). There were different degrees of colonization in colon,ileumand jejunum,the colonization rates were not more than60%.Either BALB/cCr mice or SCID/Cr mice,liver colonization were detected at3months,colonization rates in liver were between20~40%; There were different degreesof colonization in cecum,colon,ileum and jejunum in C57BL/6Cr miceinfected with H.hepaticus from1to12months, but colonization rates werelower than BALB/cCr and SCID/Cr mice.0ne mice had found colonization ofH.hepaticus in liver in C57BL/6Cr mice infected with H.hepaticus at6months, colonization rates were20%.Colonization of H.hepaticus inesophagus,stomach and pancreas tissue in different strains of mice infectedwith H.hepaticus were not detected; The colonization rates of H.hepaticus inthe digestive tract tissue in control mice were not detected.The positive rates ofH.hepaticus specific16SrRNA gene in the digestive tract tissue in differentstrains of mice infected with H.hepaticus were higher than colonization rates ofH.hepaticus.Compared with C57BL/6Cr mice,the histopathologic scores inliver,cecum and colon were more significant in BALB/cCr and SCID/Cr miceinfected with H.hepaticus (P<0.01),and Liver histopathologic scores inBALB/cCr and SCID/Cr mice infected with H.hepaticus were graduallyincreased as infection time extended within6months(P<0.05; P<0.01). Theliver histopathologic scores in,cecum and colon were no significant differenceduring6month to12month. The histopathologic scores in cecum and colon in BALB/cCr and SCID/Cr mice infected with H.hepaticus at3months werehigher than those at1months(P＜0.05；P＜0.01).The histopathologic scoresin cecum and colon were no significant difference during3month to12month.Conclusion: The colonization site of mice infected with H.hepaticus arethe lower digestive tract and liver, cecum is the site of initial colonization;Compared with C57BL/6Cr mice, histopathologic changes in liver, cecal andcolonic is more significant in BALB/c Cr and SCID/Cr mice infected withH.hepaticus, histological scores were gradually increased as infection timeextended; Different mice strains are with different susceptibility to H.hepaticus;and better H.hepaticus infection model can be obtained in H.hepaticusinoculated BALB/cCr,SCID/Cr mice. Part2Analysis of the seroprevalance H.hepaticus-IgG antibody inpatients with chronic hepatitis BObjective: To survey the seroprevalance H.hepaticus-IgG antibody inpatients with chronic hepatitis B and to analyze the possible synergisticpathogenicity of H.hepaticus in chronic hepatitis B.Methods: In this case-control study,the cases were356patients withchronic hepatitis B. All subjects were tested for the seroprevalanceH.hepaticus-IgG antibody. To compare the seroprevalance H.hepaticus-IgGantibody before and after completely absorbed H.pylori antigens. To analyzethe relation of the seroprevalance H.hepaticus-IgG antibody and severity ofdisease,sex, age and HBV loads. The controls were312sex and age matched healthy group.Results: Before absorption test, the seroprevalance H.hepaticus-IgGantibody in patients with chronic hepatitis B were still significantly higher thanhealthy controls (P<0.05);The seroprevalance H.hepaticus-IgG antibody inpatients with chronic hepatitis B and the healthy group after absorption testwere decreased than before absorption test,but the seroprevalanceH.hepaticus-IgG antibody in patients with chronic hepatitis B were stillsignificantly higher than healthy group (P<0.05). The seroprevalanceH.hepaticus-IgG antibody in patients with chronic hepatitis B and healthygroup with different sex and age were no significant difference (P>0.05);Theseroprevalance H.hepaticus-IgG antibody in patients with chronic hepatitis Bwith different HBV loads were no significant difference (P>0.05). Theseroprevalance H.hepaticus-IgG antibody were increased with Child-pughclassification raising in patients with hepatitis B cirrhosis(P<0.05).Conclusion: The seroprevalance H.hepaticus-IgG antibody were higher inPatients with chronic hepatitis B than in healthy controls；The seroprevalanceH.hepaticus-IgG antibody were increased with Child-pugh classification raisingin patients with hepatitis B cirrhosis. The seroprevalance H.hepaticus-IgGantibody had not significantly relations with different sex,age and HBV loads.H.hepaticus infection is probably linked to the progession of chronic hepatitisB. Part3Investigation of the pathogenesis ofcytolethal distending toxin in chronic liver injury inducedby Helicobacter hepaticus in different strains of laboratory miceObjective: Application of BALB/cCr, SCID/Cr and C57BL/6Cr micemice infected with Helicobacter hepaticus Standard strain ATCC51450,toobserve the pathogenesis of cytolethal distending toxin in chronic liver injuryinduced by Helicobacter hepaticus.Methods: SPF-class male BALB/cCr,SCID/Cr and C57BL/6Cr micewhich had confirmed no H.hepaticus infection were inoculated H.hepaticusstandard strain ATCC514500.2ml bacterial suspension(1×108CUF/ml),inoculated for3times with48hours intervals. Mice whichlasted for12hours were executed at1,3,6,9and12months since last afterH.hepaticus inoculation(n=5), mice liver tissue were taken for histopathologyexamiantion, H.hepaticus cdtB gene amplification and sequencing.Results: The cdtB gene were amplified in liver in BALB/cCr andSCID/Cr infected with H.hepaticus at3months. mice The cdtB geneamplification-positive rates in BALB/cCr mice were11/25(44.0%), The cdtBgene amplification-positive rates in SCID/Cr mice were12/25(48.0%),ThecdtB gene amplification-positive rates in C57BL/6Cr mice were only3/25(12.0%).Target DNAfragment of cdtB gene were about193bp,by comparingcdtB gene sequence from H.hepaticus ATCC51449,the results showed thathomology was99%; Liver tissue pathology scores with cdtB geneamplification-positive in BALB/cCr and SCID/Cr mice were significantlyhigher than the cdtB gene amplification negative mice (P<0.01).Conclusion: The cdtB gene amplification-positive rates in the liver tissue in the different strains of mice infected with H.hepaticus were amplified invarying degrees.CDT as a toxin, which may be play a pathogenic role inchronic liver injury induced by H.hepaticus.