【作者】 李红艳；

【导师】 王江滨；

【作者基本信息】 吉林大学 ， 内科学， 2012， 博士

【摘要】 第1章肝螺杆菌感染不同品系实验室小鼠模型的建立及其特征分析目的：应用H.hepaticus标准菌株ATCC51450感染不同品系实验室小鼠，观察H.hepaticus在不同消化道组织的定植状况及其病理特征，建立稳定的H.hepaticus感染实验室小鼠模型。方法：选取已经证实无H.hepaticus感染的SPF级雄性BALB/cCr、SCID/Cr、 C57BL/6Cr小鼠各30只口服灌饲H.hepaticus标准菌株ATCC51450菌液0.2毫升（1×108CUF/ml），连续3次，每次间隔48小时，对照组灌饲等量的PBS。于末次接种H.hepaticus后第1、3、6、9、12个月时，禁食12h后处死小鼠，每组5只。眼球取血后应用ELISA法检测血清H.hepaticus-IgG抗体水平；分别取小鼠食管、胃、空肠、回肠、盲肠、结肠、肝脏及胰腺组织，行病理组织学检查、微需氧菌分离培养鉴定及H.hepaticus特异性16SrRNA基因扩增。结果：BALB/cCr小鼠血清H.hepaticus-IgG抗体检出率最高，从感染后1个月至感染后12个月，在每间隔3个月的分析中，检出率均为100%；C57BL/6Cr小鼠血清H.hepaticus-IgG抗体检出率在40~60%之间；在SCID/Cr小鼠血清中无论是感染后的1个月，还是至12个月均未检测出H.hepaticus-IgG抗体。无论是BALB/cCr小鼠还是C57BL/6Cr小鼠，H.hepaticus-IgG抗体水平均在感染后6个月达最高峰，以后逐渐下降。BALB/cCr小鼠和SCID/Cr小鼠H.hepaticus感染后1个月时盲肠定植率为80%，此后持续在盲肠定植，从3至12个月盲肠定植率均为100%，此外在结肠、回肠以及空肠亦有不同程度的定植，定植率不超过60%；BALB/cCr小鼠和SCID/Cr小鼠自H.hepaticus感染后3个月起可在肝组织中发现H.hepaticus定植，BALB/cCr小鼠和SCID/Cr小鼠肝脏定植率均在20~40%之间。而C57BL/6Cr小鼠H.hepaticus感染后1至12个月盲肠、结肠、回肠、空肠亦有不同程度的定植，但定植率低于BALB/cCr小鼠和SCID/Cr小鼠；C57BL/6Cr小鼠仅在H.hepaticus感染后6个月时有1只小鼠肝组织中发现H.hepaticus定植，定植率为20%。H.hepaticus感染各品系小鼠胃、食管及胰腺组织和各PBS对照组小鼠的不同消化道组织中均未发现H.hepaticus的定植。各品系小鼠H.hepaticus感染后不同消化道组织H.hepaticus特异性16SrRNA基因阳性率高于定植率；H.hepaticus感染的BALB/cCr和SCID/Cr小鼠肝脏、盲肠及结肠病理组织学评分明显高于C57BL/6Cr小鼠（P＜0.01），BALB/cCr和SCID/Cr小鼠H.hepaticus感染后6个月内随着感染时间的延长肝脏病理组织学评分逐渐增加（P＜0.05；P＜0.01），6个月至12个月肝脏病理组织学评分有加重的趋势，但无明显差异；而BALB/cCr和SCID/Cr小鼠H.hepaticus感染后3个月盲肠及结肠病理组织学评分明显高于1个月时的组织学评分（P＜0.05；P＜0.01），3至12个月盲肠及结肠病理组织学评分无明显差异。结论：H.hepaticus感染在小鼠消化道的定植部位为下消化道及肝脏，盲肠是其起始的定植部位；BALB/cCr和SCID/Cr小鼠H.hepaticus感染后肝脏、盲肠及结肠病理组织学改变较C57BL/6Cr小鼠更为明显，病理组织学改变随着感染时间的延长而逐渐加重；不同品系小鼠对H.hepaticus的易感性不同，BALB/cCr、SCID/Cr小鼠接种H.hepaticus后可获得较好的H.hepaticus感染小鼠模型。第2章慢性乙型病毒性肝炎患者血清肝螺杆菌抗体阳性率分析目的：通过对慢性乙型病毒性肝炎患者血清H.hepaticus-IgG抗体阳性率进行调查，探讨H.hepaticus感染在慢性乙型病毒性肝炎发生发展中可能的致病作用。方法：采用病例对照研究方法对356例慢性乙型病毒性肝炎患者H.pylori抗原完全吸收前后血清H.hepaticus-IgG抗体进行检测，分析血清H.hepaticu-IgG抗体阳性率与年龄、性别、HBV载量及病程等因素之间的关系。对照组为同期性别和年龄相匹配的312例健康体检者。结果：吸收试验前慢性乙型病毒性肝炎患者血清H.hepaticus-IgG抗体阳性率明显高于健康体检者（P＜0.05）；H.pylori抗原吸收试验后慢性乙型病毒性肝炎患者及健康体检者血清H.hepaticus-IgG抗体阳性率较吸收试验前明显下降，但慢性乙型病毒性肝炎患者血清H.hepaticus-IgG抗体阳性率仍高于健康体检者（P＜0.05）。慢性乙型病毒性肝炎患者及健康体检者在不同性别及不同年龄组之间血清H.hepaticus-IgG抗体阳性率无明显差异（P＞0.05）；慢性乙型病毒性肝炎患者在不同HBV载量组之间血清H.hepaticus-IgG抗体阳性率无明显差异（P＞0.05）。随着乙型肝炎肝硬化患者Child-Pugh分级的增高其血清H.hepaticus-IgG抗体阳性率增加（P<0.05）。结论：慢性乙型病毒性肝炎患者血清H.hepaticus-IgG抗体阳性率增高，且随着乙型肝炎肝硬化患者Child-Pugh分级的增高其血清H.hepaticus-IgG抗体阳性率增加；慢性乙型病毒性肝炎患者血清H.hepaticus-IgG抗体阳性率与年龄、性别及不同HBV载量之间无明显关系；H.hepaticus感染可能与慢性乙型肝炎的发生发展相关。第3章细胞致死性膨胀毒素在肝螺杆菌感染不同品系实验室小鼠慢性肝损伤中致病作用探讨目的：应用H.hepaticus标准菌株ATCC51450感染不同品系实验室小鼠，观察细胞致死性膨胀毒素在H.hepaticus诱导的肝脏病理损伤中的致病作用。方法：选取已经证实无H.hepaticus感染的SPF级雄性BALB/cCr、SCID/Cr及C57BL/6Cr小鼠各30只，口服灌饲H.hepaticus标准菌株ATCC51450菌液0.2毫升（1×108CUF/ml），连续3次，每次间隔48小时。于最后一次接种H.hepaticus后1、3、6、9、12个月时，禁食12h后处死小鼠，每组5只。随机留取各组小鼠的肝脏组织分为2份，分别行H.hepaticuscdtB基因扩增并测序及病理组织学检查。结果：BALB/cCr和SCID/Cr小鼠H.hepaticus感染后3个月时肝组织中扩增出cdtB基因，BALB/cCr小鼠肝组织cdtB基因阳性率为11/25（44%）；SCID/Cr小鼠肝组织cdtB基因阳性率为12/25（48%）；C57BL/6Cr小鼠H.hepaticus感染后肝组织cdtB基因阳性率仅为3/25（12%）。cdtB基因目的片段约为193bp，PCR产物测序结果与genebank上H.hepaticusATCC51449cdtB基因序列比较同源性为99%；BALB/cCr、SCID/Cr小鼠H.hepaticus感染后cdtB基因阳性者的肝脏病理组织评分明显高于cdtB基因阴性者（P＜0.01）。结论：H.hepaticus感染不同品系小鼠肝组织中不同程度地扩增出H.hepaticus cdtB基因， CDT作为H.hepaticus分泌的一种毒素，可能在H.hepaticus诱导的慢性肝损伤中发挥一定的致病作用。更多还原

【Abstract】 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.更多还原