【作者】 周建嫦；

【导师】 徐采朴； 张建中；

【作者基本信息】 第三军医大学 ， 内科学， 2001， 博士

【摘要】 目前，幽门螺杆菌（Helicobacter pylori，H pylori，Hp）菌型差异导致感染结局不同已得到普遍共识。研究显示，多数欧美国家CagA阳性菌株感染与非贲门部胃癌和十二指肠溃疡发生相关，而亚洲地区菌株的CagA阳性率很高，且在不同胃肠疾病间的分布无差异。我国相关的研究结果很不一致。由于cagA/CagA具有显著的多态性，不同菌株cagA基因序列、CagA分子大小、抗原性及磷酸化修饰间的差异，可导致其致病性不同。日本、德国、南非的研究显示存在与胃癌或消化性溃疡相关的cagA/CagA亚型。由此可见，各种cagA/CagA亚型菌株在各地分布不同，可能是造成各地研究结果不一致的重要原因，而检测手段敏感性的差异，也可能导致研究结果的偏倚。为了明确cagA/CagA及其亚型在我国Hp菌株中的分布情况，以及有无与疾病相关的特定cagA/CagA亚型，我们进行了如下研究。 1、通过PCR扩增cagA基因，结合SDS-PAGE检测CagA蛋白表达的结果，分析我国四省市不同疾病分离株cagA/CagA的分布情况。 2、采用免疫印迹杂交的方法，分析38株国内Hp菌株及2株国际标准株的CagA抗原对三种不同菌株兔血清的反应性，比较CagA抗原性多态性在不同地区及胃十二指肠疾病的分布差异。 3、采用PCR方法分析77株国内Hp菌株cagA基因的3’端可变区变异及其与胃十二指肠疾病发生的关系。 4、克隆4株背景清楚的Hp菌株（国际标准菌株SS1和国内2株胃癌分离株和1株十二指肠溃疡分离株）的全长cagA基因，测序后与GenBank中已知的8个cagA基因核苷酸序列和推定氨基酸序列进行了同源性分析；分析了它们之间的磷酸化位点差异 5、构建了上述4株菌的CapA基因的重组原核表达质粒。 我们的结果显示：l）我国菌株的 cagA儿agA阳性率极高，在不同地区 的分布无差别，与疾病发生无显著相关，因此，cagA／CagA不能作为我国 Hp的毒力标志。2）我国菌株 CagA的抗原性具有显著多态性，CagA抗原 性与致病性存在一定的关系，与抗 NCTC 63 7的 CagA抗原性相近的 CagA，可能与消化性溃疡关系较为密切。检测CagA抗原或抗体时，应联 合使用至少三种抗 CagA抗体或 CagA抗原。3）根据 3’端可变区长度，我。 国Hp菌株的cagA基因至少可分为l、11、Ill三型，不同地区、不同疾 病来源菌株的cagA基因均以1型为主，11型和皿型cagA基因阳性菌株感 染是否与临床结局有特殊关联，有必要对目前病例进行随访观察和更大规 模的人群调查研究。4）所测的国际标准菌株％ 和国内2株胃癌分离株和 1株十二指肠溃疡分离株的全长cagA基因序列已登录GenBank，序列号分 别为AF249275、AF247651、AF367250、AF367251。同源性分析表明，我 国菌株与日本菌株的cagA基因核音酸序列及推定CagA氨基酸序列间聚类 关系明显，但 GenBank中 12株 Hp菌间未发现明显的疾病聚类关系。CagA 磷酸化位点不同可能与临床感染结局间存在某种联系，但需进一步研究明 确。5）获得了上述 4株 Hp菌株 CapA融合蛋白的重组表达质粒，为进一 步进行CagA的功能和致病性研究和人群血清学调查研究奠定了基础。更多还原

【Abstract】 Numerous previous studies in Western countries have highlighted some Helicobacter pylon (H pylon) strains are more pathogenic than others, and that CagA-positive If pylon strains are associated with severe gastroduodenal diseases. However, this is not the case in Asian countries where there are high prevalence of cagA-positive strains infection. Those studies conducted in China had revealed conflicting results.The paradox that CagA-positive H. pylon infection correlates to two mutually exclusive diseases: gastric cancer and duodenal ulcer, and results in various clinical outcomes in different populations, may be due to the heterogeneity of cagA/CagA, which had been suggested by studies conducted in Japan, Germany and South Africa. Discriminatory techniques detecting cagA or CagA employed in different studies may be another contributory factor. Recent data have suggested that the tyrosine phosphorylation of CagA may also play a role.cagA/CagA is highly divergent, either at gene level or protein level, which is most marked in the 3?region where there are variable number of repeat sequences. Previous studies showed that CagA protein in larger size and with stronger antigen are more likely to correlate to gastric adenocarcinoma.Therefore, we made an attempt to examine the heterogeneity of cagA/CagA of Chinese H. pylon isolates, and determine whether there are distinct cagA/CagA types circulating in different areas in China, so as to character gastroduodenal diseases-specific cagAlCagA types.The prevalence of cagAlCagA of Chinese H. pylon strains was firstdetermined by detecting the cagA gene using polymerase chain reaction andCagA protein using SDS-PAGE simu1taneously, Which revealed very high. Sothe anigenic heterogeneity of CagA of 38 strains was then deterrnined byaligning and comparing their reactivity to thIee different rabbit sera immunizedby H Pylori strains CAPM N62, NCTCl1637 and CAPM Z-2 respectivelyusing western blots. To examine whether distinct cagA alleles may circulate indifferent areas and correlate to different clinical outcomes, the variablesegment of 3’ region of cagA gene of different clinical origin were amplifiedby PCR, and the length of PCR products were compared. In order to fullyunderstand the relationship between heterogeneity of CagA and clinicalpresents, the full-length cagA genes of two gastric cancer-associated and oneduodena1 ulcer-associated H PylOri strains and strain SSl were cloned andsequenced, and the homology of their nucleotide and deduced amino acidsequences as wel1 as phosphorylation motifS preseni in CagA were analyzed.Considering it is difficult to get 1ots of CagA from H Pylori culture for studiesof the function and pathogenesis of CagA, cagA of these fOur strains werecloned inio PinPointTM Xa-l T-vector to constructed recombinant plasmidsexpressing CagA fused to PinPointTM biotinylation segment.Our resu1ts showed: (l) There is a high prevalence of cagAjCagA ofChinese H Pylori isolates irrelevant geograPhic or clinical origin, Which agreeswell with those of other Asian couniTies, and suggests that cagA/CagA is notthe virulent mark of H Pylori strains in China. (2) None of the threeimmunized sera cou1d recognize CagA of all isolates, howeveT, each CagAcould be recognized by at least one serum. The antigenic heterogeneity ofCagA was grouPed as I -V, group II which reacted most strong1y withserum immunized by NCTC11637 was more likely but not significantlycorrelated to peptic ulcer’ (3) cagA genes were designed to three types I, II,and III according to the amplified PCR product length of variable segment of3’ region, of which was about 825bp, 900bp or 950bp respectively. Type IcagA gene predominates in China, both the rare two tyPes II and Ill cagAgene present in only two iso1ates respectively. (4) The complete sequences ofIVcagA genes of strain SS1, CAPM N62, CAPM N93 and CAPM Nih have been deposited in GenBank under accession number AF247651, AF249275, AF36更多还原