摘要
幽门螺杆菌(Hp)是一种世界性流行的革兰氏阴性细菌，Hp的人群感染率约20.80％，是十二指肠溃疡和慢性活动性胃炎的主要致病因素之一，与胃癌的发生有一定的关系。由此吸引了众多学者对Hp的广泛深入的研究，但仍有许多问题有待解决，如口腔是不是Hp的寄居环境之一；为何Hp感染后，临床结局和转归有很大差异；如何判断Hp根治性治疗失败后是属再感染抑或复燃等。故本研究同时采用16sRNA和vacA为模板设计的引物，对唾液和胃粘膜中的Hp直接进行扩增，并与常规Hp检测法--快速尿素酶(RUT)和组织学银染法进行比较，观察其特异性和灵敏度。由于采用了一对以vacA基因的特殊部位为模板设计的引物，可将Hp扩增出259bp、286bp两种产物，而每一种Hp只能扩增出一种片段，前者来源于可表达VacA蛋白的60190菌株的信号序列，称为vacA_1；后者来源于不表达VacA蛋白的Tx30a菌株的信号序列，称为vacA_2。同时，对Hp的vacA PCR产物进行单链构象多态性分析(PCR-SSCP)，期望用Hp的毒力因子(vacA)，将不同Hp区分开来，寻找与临床某一疾病相关的细菌DNA单链构象。另外，根据PCR-SSCP分型结果，对3例DU的PCR产物行基因序列分析，观察是否有某处或某几处vacA基因有点突变或缺失及其与临床疾病的关系。并利用Southern杂交来证实PCR产物的特异性。此外，利用蛋白印迹法(western blot)来判断Hp的vacA和cagA基因型与表现型间的关系，以期建立一种简单、实用的临床诊断法来初步判断Hp的毒力强弱。最后，采用原位凋亡检测法和电镜分析，对Hp感染的胃十二指肠疾病进行研究，来探讨细胞凋亡与Hp感染在胃十二指肠疾病中的意义。本文结果及意义如下：
1．本文利用16sRNA和vacA为模板设计的引物对149例Hp阳性患者的胃粘膜直接行PCR检测，其特异性和灵敏度均与临床常规的Hp诊断法
Helicobacter pylori (H.pylori)organisms are gram-negative bacteria infected the gastric mucosa of 20-80% of human population throughout the world. Most infected persons are asymptomatic ,only a few develop serious gastroduodenal diseases. It is a major caustive agent of duodenal ulcer and chronic gastritis, and it is possibly related with the devolopment of gastric cancinoma. Thus, it attracts a lot of investigators focusing on the further and wider study of H.pylori. However, many fields regarding the pathogenesis, transmission and management of the disease are not yet well-understood. For example, whether oral cavity is one of the colonizing enviroment of H.pylori;why there are great difference in clinical outcome after infection of H.pylori; how to distinguish between recrudescence from reinfection after H.pylori eradication. So we examined the usefulness of 16sRNA and vacA gene to simutaneously amplify H.pylori in saliva and gastric tissues, and compared its specificity and sensitivity with conventional detection approach of H.pylori ( the rapid urease test and Warthin-Starry test. Using the specific primers amplifying and differentiating the typeS_1 and typeS_2 signal sequences, it was predicted that 259-and 289-bp products would be PCR-amplified products from typeSi and typeS_2 strains, respectively. Each strain only yield a PCR product of one of the two. The former size derived from the signal sequences of Tox~+60190 expressing cytotoxin, named vacA_1; the latter size oringinated from the signal sequences of Tox" Tx30a unexpressing cytotoxin, called vacA_2. In addition, PCR-SSCP analysis were performed with PCR products of vacA, it was predicted to differeniate various H.pylori strains and explore specific subtypes of H.pylori strains for their association with clinical
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