【作者】 杨永利；

【导师】 段广才；

【作者基本信息】 郑州大学 ， 流行病与卫生统计学， 2002， 硕士

【摘要】 幽门螺杆菌(Helicobacter pylori，H.pylori)是世界上最常见的病原菌之一，是慢性B型胃炎和消化性溃疡的重要致病因子，也与胃癌和粘膜相关样淋巴组织淋巴瘤有一定的关系。1994年，世界卫生组织(WHO)正式将H.pylori列为人类的第一类致癌剂(Group I carcinogen)，美国疾病预防和控制中心也将H.pylori感染性疾病列为近年新出现的传染病。 但在H.pylori的研究领域中，有些问题至今仍未阐明，如H.pylori在自然人群中的感染率近60％，但有近20～30％的感染者可终身携带H.pylori而不发病；50％以上的感染者仅有不同程度的慢性炎症；10～15％可发生消化性溃疡；只有极少数发生胃部恶性肿瘤。这些不同的疾病结局是否与基因型别有关，目前尚未取得一致意见。造成此领域知识缺乏的主要原因是没有一套合适的鉴别方法。 为广哎才学么一子I生竿虏fM02届J颀士毕业殷文 砌刀氟q劳带丢墓历HX’xrvn分折J致膨仪死史 基于Hpylori表型特征的一致性及基因组结构高度变异 性的特点，本研究应用PCREFLP方法，对临床分离的46株Hylori（其中溃疡株2 5例，非溃疡株刀例）的鞭毛基因 ＊agellin gene，Fa gene）进行分型，旨在探讨鞭毛基因的变异性及鞭毛基因分型在溃疡组和非溃疡组的分布有无差别，为Hpylori的鉴别提供参考依据。 方法 1 幽门螺杆菌的分离培养：内镜下用常规方法取胃肠疾病思者胃粘膜组织块，涂抹法接种于改良布氏培养基上，37OC恒温培养箱微需氧环境培养5刁大，对可疑菌落进行菌落形态、石炭酸复红染色观察和生化鉴定，取阳性者传代。菌不保存于－80 OC。 2 HPyloriW基因的PCR－RFLP分析：PCR扩增46株HPylori（其中溃疡株 25例，非溃疡株刀例）侧基因中间区域，1．5％琼脂糖凝胶电泳鉴定PCR扩增产物，970hp位置有一条带者进一步用 Hpall和 Hindlll内切酶消化，2刀％琼脂糖凝胶电泳分离后得到尸d基因PCR－RFLP谱型，记录结果，进行聚类分祈。 3 Hpylori M基因的 PCRKFLP分析：PCR扩增 46株HPylOOI（其中溃疡株25例，非溃疡株ZI例）加B基因中间区域，1．5％琼脂糖凝胶电泳鉴定PCR扩增产物，930hp位置有一条带者进一步用 Hpa 11和 Hindlll内切酶消化，2刀％琼脂 2斯W大算J生学贸C刃肥居）旋士毕业极艾 幽门燎抒菌般丢三历PC尸rkYIP分行及纹厉烂研究糖凝胶电泳分离后得到人aB基因PCR｝FLP谱型，记录结果，进行聚类分析。 结果 ljlaA基因 PCR－RFLP分祈：46株 Hpylori iaA基因经PCR扩增后，产物长度为970hp，进一步将PCR产物用Hpa11酶切后，产生4种酶切图谱：Hilldlll酶切后产生3种酶切图谱，将j7aA基因 Hpall和 Hindlll酶切图谱进行编码并用统计学软件进行聚类分祈，可将仰基因分为三型（I型、11型和Ill型），三型在溃疡组和非溃疡组的构成比无差别（尸＝0．294）。 2 iaB基因 PCR－RFLP分析：Hpylori iaB基因 PCR 扩增产物长度为 930hp，Hpa 11酶切后，仅有 l种酶切图 谱：HilldIJI酶切后有 2种酶切图谱，综和尸。B基因 Hps 11 和 Hindlll酶切图谱，可将 M基因分为两型（1型和 11 型），二型在溃疡组和非溃疡组的构成比也无差别 （尸＝0．658）。 3j7aA和加B基因综合分型结果：将j7aA和j7aB基因的fLP图谱编码并输入SPSS10刀软件进行聚类分祈，选择合适的分界点，46株 Hpyloj’l可分为三型（I型、11型和Ill型），三型在溃疡组和非溃疡组的构成比仍无差别0二0．294）。 结论 3郑训丈学牙＃J主学裙c刃02屠J＠士毕业活文 幽／刀揖年菌鞭宅募毋尸口尸1W℃尸分折上反厉注研灭 1 我国临床分离的 Hpylorij7a基因比较保守，绝大多数菌株具有的相同 Hpa 11和 Hindlll酶切图谱。 2首次显示国内 Hpylori加基因 Hpa 11和 Hindlll主纹图谱示意图，作为一种标准方法建立的标志，为今后j7a基因PCR－RFLP分析提供参考依据。 3 首次运用统计学软件进行聚类分析，将我国46株临床分离的Hpyloride基因分为三型。 4 初步证明了加基因型别与不同的胃十二指肠疾病无关，这为临床上 Hpytori疾病相关株和非疾病相关株的鉴别提供了参考依据。建议今后Hpylori的鉴别应重在比较疾病相关株和非疾病相关株之间的差别，而不应仅局限于不同胃十二指肠疾病结局之间的比较。更多还原

【Abstract】 Helicobacter pylori (H.pylori) is one of the most common bacterial pathogens of human beings. Many studies have shown that it can cause chronic type B gastritis and peptic ulcers, and it is also related to the gastric carcinoma and the gastric mucosa-associated lymphoid tissue (MALT) lymphomas. In 1994, World Health Organization (WHO) classified H.pylori as a definite (class I) carcinogen. Centers for Disease Control and Prevention (CDC) have classified H.pylori infectious diseases as one of the new emerging infectious diseases.The infection rate of H.pylori is almost 60% in the population. However, not all the infected individuals would have gastrointestinal diseases. 20-30% of the infected persons are asymptomatic, 50% may have gastritis, 10-15% may have pepticulcers, a proportion of them may develop gastric carcinoma. This suggests that different genotypes of H.pylori might cause different diseases. The mechanism of pathogenic diversity and the mode of transmission are not clear at present. The pancity of knowledge in these areas is in part due to the lack of a reliable universal classification method for H.pylori.Because H.pylori is one kind of bacteria which have similar appearance, but have high genetic diversity. In this study, PCR-RFLP technique was used to classify 46 clinical isolates of H.pylori (25 ulcerous strains and 21 non-ulcerous strains). The relationships between the genotypes and different diseases were also analyzed.Although the exact pathogenic mechanism is unclear, one point is very obvious. H.pylori must colonize in the stomach mucus firstly, can then play pathogenic role further. In the gnotobiotic piglet model, the mutant strains of fla gene or aflagellate strains can not colonize in the mucus, so lose pathogenic ability. Studies also show that the strains with stronger motility have stronger pathogenic ability. Now, we have known the flagellar filament contains two main flagellins, the major flagellin, FlaA (53.2kDa) and a minor flagellin, FlaB (53.9kDa).They are coded by flaA gene (1533bp) andflaB gene (1597bp) respectively. The two genes have been cloned and sequenced. Methods1 Isolation and identification of H.pylori: Gastric mucosal biopsy specimens were obtained with routine endoscopy, and daubed on the medium. After incubation at 37 癈 in micro-aerophilic conditions for 5 to 7 days, suspicious colonies were distinguished by typical colonial morphology, straining method, and biochemical reactions. The positive colonies were subcultured and kept at -80C.2 PCR-RFLP of flaA gene: The middle region of flaA gene was amplified by PCR method. PCR products of 970bp were obtained, and the products were digested with restriction endonuclease Hpa II or Hind III. The bands of PCR-RFLP patterns were coded and analyzed by cluster analysis method with the use of SPSS 10.0 software.3 PCR-RFLP offlaB gene: The middle region offlaB gene was amplified by PCR methods. Products of 930bp were obtained. Then PCR products were digested with restriction endonuclease Hpa II or Hindlll. The bands of PCR-RFLP patterns were coded and analyzed by cluster analysis method with the use of SPSS 10.0 software.Results1 PCR-RFLP offlaA gene: There were 4 kinds of restrictionpatterns after PCR products offlaA gene for 46 strains ofH.pylori were digested with Hpall, and 3 kinds of patterns with Hindlll. 46 strains were classified into three genotypes by cluster analysis. There were no relationships between genotypes and different diseases (P=0.294).2 PCR-RFLP offlaB gene: There was only one restriction pattern after PCR products offlaB gene for 46 strains of H.pylori were digested with Hpa II, 2 kinds of patterns with Hindlll. 46 strains were classified into two genotypes by cluster analysis. There were no relationships between genotypes and different diseases (7M).658).3 The comprehensive result of the two genes: If the genotypes of two genes were put togather, 46 clinical strains were classified into three genotypes by cl更多还原