【作者】 陈国弟；

【导师】 吴勤动； 赵昌峻；

【作者基本信息】 浙江大学 ， 内科学（消化系病）， 2001， 硕士

【摘要】 自从1982年Warren和Marshall从慢性活动性胃炎患者的胃粘膜中分离出幽门螺杆菌（Helicobacter pylori，H.pylori）以来，H.pylori受到了极大的关注。现在认为H.pylori是慢性胃炎的主要致病因素，与消化性溃疡、胃癌及胃粘膜相关性淋巴样组织淋巴瘤的发生发展关系密切，WHO和国际抗癌联盟已将H.pylori列为Ⅰ类致癌物。H.pyori还可以通过慢性持续性的感染，引起免疫反应，与胃肠外多种疾病有关，如冠心病、急性心肌梗塞、儿童生长迟缓及格林巴利综合征等。 H.pylori的人群感染率高，推测全球近一半人感染过该菌。H.pylori在人体感染时间长达数十年，甚至有些人终生携带该菌。疫苗接种将是预防H.pylori感染最有效的方法。兔疫研究初期使用的是H.pylori的超声粉碎抗原，粗制抗原含抗原成分较多，存在交叉免疫反应，副反应较大。最近报道了脂多糖、粘附素、热休克蛋白、全菌抗原、尿素酶、空泡毒素（VacA）和毒素相关蛋白（CagA）及过氧化氢酶等疫苗抗原可诱导保护性抗体的产生，但各种抗原组分都有优缺点。目前研究最多的是尿素酶抗原，尤其是尿素酶B亚单位（Ure B）的特异性更受重视。迄今国内外虽已有多篇研究报道，但目前尿素酶疫苗的研究仍存在一些问题，如H.pylori菌株的多样性增加了开发抗H.pylori疫苗的难度。我们根据ure B的保守序列，人工合成了Ure B核心多肽，以克服H.pylori菌株尿素 2001浙江大学硕士学位论文酶的变异性。尿素酶ureA、ureB是结构基因，但为了避兔H．pylon尿素酶变异株的影响，本课题应用国际标准株、临床分离株及尿素酶突变株混合菌液制备尿素酶（全长）蛋白，用以制备多克隆lgG抗体，建立血清游离H．Pylon UreB及UreBtsG、UreB－IgA、UreBtsM三型兔疫复合物的酶免疫测定法。以验证H．pylon阳性患者血清中是否存在 Ure B，讨论 Ure B能否引起特异性的免疫应答以及免疫应答的形式，探讨 Ure B核心多肽作为疫苗抗原的可能。 142例临床收集的患者，经1999年H．PylOOi阳性科研诊断标准（H．PylOOiIgG、RUT或UBT、MB染色，三项中任二项阳性）诊断，其中61例H．pylon阳性。61例阳性患者血清检测结果进行统计学分析。本研究血清游离 UreB阳性率63．9％，UreB－IgG阳性率62．3O，UreB－IgA阳性率75．4O，UreB－IgM阳性率78．7％，四项联检任一项阳性有60例，阳性率为98。4％。联检阳性率显著高于单项的阳性率。而在 39例游离 UreB阳性的血清中，UreBtsG阳性率刀石％，UreB伦A阳性率74．4％，UreBteM阳性率76．9％，三项免疫复合物联检任一项阳性有37例，阳性率94．9％。联检阳性率显著高于单项的阳性率。进一步分析发现血清与 UreB－IgG型复合物之间的相关性有统计学意义，而游离 Ure B与UreBtsA型、UreB－IgM型复合物以及三型免疫复合物之间的相关性均无统计学意义。61例血清四种形式 UreB的检测结果见表 1。 表 161例 H．pylon阳性患者血清游离 UreB及 UreB－IgG、UreB－IgA、 UreB伦咖三型免疫复合物检测结果 方法 阳性 阴性 游离 UreB 39 22 UreB－IgG＊38 23 UreB－IgA 46 UreB－IgM 48 13 一 2一 2001浙江大学硕士学位论文基于以上工作，可得到如下结论： 1．建立了血清 H．pylon UreB及其兔疫复合物的酶免疫测定法。 2．H．pylon感染患者血清中存在游离UreB，UreB能引起特异性兔疫应答， 并能形成特异性免疫复合物。不同患者所引发的免疫应答类型不同，形 式有 IgG型、IgA型及 IgM型等； 3．血清游离UreB与UreB－IgG型免疫复合物相关，而游离UreB与 UreBtsA、UreB－IgM型兔疫复合物以及三型免疫复合物之间均无相关 性； 4·H．pylofi UffB核心多肽具有特异性的抗原表位，从而为 UreB核心多肽 作为H．pylon疫苗抗原提供了理论依据。更多还原

【Abstract】 Helicobacter pylon (H.pylori) has been attracted more attention since it was separated from gastric mucosa and cultured successfully by Marshall and Warren in 1982. 1-Lpyloni is a most important etiological factor of chronic gastnitis and close association with peptic ulcer. It has also near relationship between l-Lpylori and gastric carcinoma (or Mucosa Association Lymphoid Tissue Lymphoma). In 1994, IARC listed H.pylori as Grade I Carcinogen. hi recently, it has been indicated that coronary heart disease, acute myocardial infarction, child growth retardation and Guillain-Barre syndrome is also close association with infection of H.pyloni. H.pylori is a worldwide epidemic bacteria. There are aboute half peoples infected H.pylori. H.pyloni can exist in humam body for several decades, even as long as human life. It is very important to research -4--- 2001 ~ vaccine to prophylaxis the infection of H.pylori. On the early stage, the antigen is the ultrasound fragmenting components of outer membrane. The ultrasound fragmenting antigen contain more components, so there exists crossing response and adverse effect. Later, the vaccine antigens is experienced for lipopolysaccharide, adhesin, heat shock protein, urease, VacA, CagA and catalase. Each antigen has its virtues and shortcomings. I-l.pylori are located in the stomach mucosa and the component of outer membrane can be absorbed into the?circulation and invoke the immune response. So there are component of outer membrane in serum. Little studies have been carried out for detecting H.pylori antigen in serum. In recent, some literatures indicate that there is presenting 1-I.pylori soluble antigen and antigen-antibody complex in serum from patients with H.pylori infection. Previous antigen for vaccinating animal is H.pylori general component in outer membrane,it is crude and has a lower specificity. Now more and more scholars are focused on the urease antigen. H.pylori is featured for high activity urease, The active part of urease is locating outer membrane. Being vaccinated antigen, the recombination urease subunit A and B can protect 60%-80% mouse from infecting H.felis, but subunit B is better than subunit A. It hints that subunit B is a highly specific antigen. But there are also more difficulties in the research of urease antigen, it is marked for the strain diversity of H.pylori. So we synthesis the UreB core polypeptide according to H.pylori ureB conserved sequence. We Vaccinated caprine with UreB core polypeptide for inducing polyclonal IgG antibody. An enzyme-immunoassay is created to detect H.pylori free UreB and its immune complexes in serum. We want -5? 2001 ~ to verificate if there exists UreB in serum from patients with H.pylori infection, and discuss the relationship among H.pylori free UreB and its immune complexes in serum. Thereby we are to provide some theoretic for UreB core peptide as vaccine antigen. All patients are diagnosed by H.pylori IgG, Rapid urease test, MB Stain and Urea Breath test ( any two positive of IgG, Rapid urease test or Urea Breath test and MB Stain is diagnosed as positive). There are 61 patients infected H.pylori among 142 clected patients. In this study, we find the positive rate of fr更多还原