不同消化性疾病来源的幽门螺杆菌毒力基因cagA、vacA、oipA、dupA的检测及其意义

发布时间：2018-01-09 23:07

本文关键词：不同消化性疾病来源的幽门螺杆菌毒力基因cagA、vacA、oipA、dupA的检测及其意义　出处：《福建医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：[背景和目的] 幽门螺杆菌（Helicobacter pylori，以下简称Hp）是引起胃炎、消化性溃疡、胃粘膜淋巴组织相关性淋巴瘤和胃癌的重要病原体。全球约有一半以上人口感染Hp，但在感染人群中只有约20%发生消化性疾病，且疾病的严重程度不同，其原因尚未阐明，可能与细菌基因高度多态性、宿主和环境等因素有关。Hp cagA、vacA、oipA、dupA四种毒力基因是幽门螺杆菌致病的重要因素。幽门螺杆菌感染的特点，主要体现在菌株基因多态性和地区分布差异性，且各种基因与疾病之间的关系也有地区差异性。本研究旨在调查胃癌高发的福州地区的Hp cagA及其C端的EPIYA基序、vacA和dupA分型以及oipA基因开关状态，了解本地区的幽门螺杆菌基因多态性，并探讨本地区消化性疾病与这些基因、性别和年龄之间的关系，以便对幽门螺杆菌的致病机制有更加深入的认识。 [材料和方法] 1．幽门螺杆菌的分离与鉴定：从福建省肿瘤医院，福建医科大学附属协和医院和福建省立医院采集的胃组织标本（来自慢性胃炎、消化性溃疡和胃癌患者），分离培养Hp，组织研磨后接种于布氏平板，于厌氧培养箱培养3-5d，经尿素酶、过氧化氢酶及形态学鉴定确认。 2．用高纯度基因组DNA模板提取试剂盒（购自罗氏公司）提取Hp基因组DNA，并以其为模板，，PCR扩增cagA3′端序列、vacA基因s区、i区、m区、oipA信号区、dupA（jhp917和jhp918）基因。 3． cagA3′端和oipA信号区PCR后送生工生物（上海）有限公司，sanger法，ABI-PRISM3730测序仪进行基因测序，Bioedit分析软件分析EPIYA基序类型和oipA基因开关状态。 4．统计分析：运用方差分析比较各疾病组之间年龄的差异，卡方检验分析疾病与各基因之间的关系，logistic回归分析各疾病组之间的基因差异。 [结果] 1.分离培养获得82株幽门螺杆菌，其中分离自慢性胃炎患者37株，消化性溃疡患者25株（其中胃溃疡12株，十二指肠溃疡13株），胃癌患者20株。疾病与性别和年龄的关系：单因素卡方分析胃炎，消化性溃疡和胃癌组间性别差异，χ~2=7.39，P0.05，P=0.022，差异有统计学意义，表明消化性溃疡和胃癌患者中男性比例高于胃炎患者中男性的比例；方差分析三组疾病间年龄差异，F=1.51，P0.05，P=0.227，差异无统计学意义。 2.基因检测及统计学分析结果：所有被测菌cagA基因均为阳性， EPIYA分型结果：胃炎菌株：EPIYA-ABD32株（86.5%），EPIYA-AD1株（2.7%），EPIYA-ABBD4株（10.8%）；消化性溃疡菌株： EPIYA-ABD23株（92%），EPIYA-AB1株（4%），EPIYA-ABBD1株（4%）；胃癌菌株：EPIYA-ABD19株（95%），EPIYA-ABBD1株（5%）, EPIYA-ABD和非EPIYA-ABD型三组疾病间卡方分析，χ2=1.20，P0.05P=0.639。总体菌株，EPIYA-ABD型74株，占90.2%，表明本地区，EPIYA分型以东方型的EPIYA-ABD为主，各疾病组之间无显著差异。 vacA基因分型结果：胃炎菌株：s1c/i1/m1b13株(35.1%)，s1c/i1/m224株（64.9%）；消化性溃疡菌株：s1a/i1/m1b1株（4%），s1c/i1/m1b15株（60%），s1c/i1/m29株（36%）；胃癌菌株：s1c/i1/m1a1株（5%），s1c/i1/m1b9株（45%），s1c/i1/m210株（50%）。未检出s2型、i2型vacA。vacA m区，m2型菌株所占比例为52.4%，m1型所占比例为47.6%。logistic回归分析，消化性溃疡组与胃炎组间m1型相比，OR=3.282，95%可信区间为（1.138，9.468），P0.05P=0.028，差异有统计学意义，表明vacA m1型增加了患者发展为消化性溃疡的危险性；m2型菌株比例高于文献报道的东亚地区的韩国（8.65%），低于文献报道的东南亚地区的越南（63.8%）。所有被测菌oipA基因均为阳性，测序分析其开关状态均为“开”（均为功能性oipA），“CT”重复序列特征有“none”（6株），“1+4”（2株），“2+1”（2株），“2+1+1+1”（4株），“3+1”（56株），“3+2”（5株），“5+2”（1株），“6”（5株），“3”（1株）九种类型。 dupA基因阳性率，慢性胃炎为11株（29.7%），消化性溃疡5株（20%）（其中胃溃疡3株，十二指肠溃疡2株），胃癌9株（45%）。十二指肠溃疡组与非十二指肠溃疡组间dupA基因卡方检验，χ~2=0.76，P0.05P=0.382，差异无统计学意义。表明作为十二指肠溃疡重要指标的DupA基因在本研究中，未体现这一重要作用。表明dupA基因尚不能作为福州地区Hp致十二指肠溃疡的标记基因。综合分析EPIYA、vacA和dupA基因，EPIYA-ABD+dupA+s1/m1菌株在各疾病组间,胃炎菌株中占5.4%，消化性溃疡菌株中占16%，胃癌菌株中占30%，卡方检验χ~2=6.340，P0.05P=0.036，表明EPIYA-ABD型，vacA基因s1/m1型，dupA同时阳性菌株增加了患消化性溃疡和胃癌的危险性。 [结论] 1.福州地区Hp菌株的cagA EPIYA分型体现了东方国家Hp菌株的特征，以EPIYA-ABD型为主，未见西方型EPIYA-ABC菌株。 2. vacA m1型是消化性溃疡的危险因素，vacA基因m2型所占比例高。远比在m区中以m1b型为主的韩国要高，但比东南亚的越南的要低。 3.尚不能认为Hp菌株dupA基因可以作为福州地区Hp致十二指肠溃疡疾病的标记基因。 4.EPIYA-ABD型、vacA基因s1/m1型和dupA三者同时阳性的菌株增加了患消化性溃疡和胃癌的危险性。
[Abstract]:[background and purpose]
Helicobacter pylori (Helicobacter pylori, hereinafter referred to as Hp) is the cause of gastritis, peptic ulcer, gastric mucosa associated lymphoid tissue is an important pathogen of lymphoma and gastric cancer. About half of the world population is infected with Hp, but only about 20% people infected in the occurrence of peptic disease severity and disease, the reason has not been elucidated may, and the bacterial gene is highly polymorphic, host and environmental factors such as.Hp cagA, vacA, oipA, dupA four kinds of virulence genes is an important pathogenic factors of Helicobacter pylori. Helicobacter pylori infection characteristics, mainly reflected in the differences between strains gene polymorphism and region distribution, and the relationship between genes and diseases there are also regional differences. Hp cagA and C EPIYA motif in the Fuzhou area this study aimed to investigate the high incidence of gastric cancer of the end, vacA and dupA type of oipA gene and switch state solution in the region. Gene polymorphism of Helicobacter pylori and the relationship between digestive diseases and these genes, sex and age were discussed in order to have a deeper understanding of the pathogenesis of Helicobacter pylori.
[materials and methods]
Isolation and identification of 1. Helicobacter pylori: from the tumor hospital of Fujian Province, Affiliated Hospital of Fujian Medical University and Fujian Province-owned Hospital collected gastric tissue samples (from chronic gastritis, peptic ulcer and gastric cancer), isolated and cultured Hp tissue grinding after vaccination in Brandt tablet, in anaerobic incubator 3-5d, confirmed by urease, catalase and the morphological identification.
2., we extracted genomic Hp from genomic DNA by using high purity genomic DNA template extraction kit (purchased from Roche) and used PCR as template to amplify cagA3 'end sequence, vacA gene s region, I region, m region, oipA signal region, dupA (dupA and DNA) gene.
Gene sequencing was carried out on 3.cagA3 'end and oipA signal area PCR evacuation bioengineering Biology (Shanghai) Limited company, Sanger method and ABI-PRISM3730 sequencing instrument. Bioedit analysis software was used to analyze EPIYA motif type and oipA gene switch state.
4. statistical analysis: variance analysis was used to compare age differences among different disease groups. Chi square test was used to analyze the relationship between diseases and genes. Logistic regression analysis was used to analyze the genetic differences among disease groups.
[results]
82 strains of Helicobacter pylori isolated from 1. patients with chronic gastritis, including 37 strains isolated from patients with peptic ulcer, 25 strains (12 strains of the gastric ulcer, duodenal ulcer and 13 strains, 20 strains) in patients with gastric cancer. The relationship between disease and age and sex: Chi square analysis of gastritis, peptic ulcer and gastric cancer group the difference between gender, X ~2=7.39, P0.05, P=0.022, the difference was statistically significant, showed that the proportion of male peptic ulcer and gastric cancer were higher than that of male patients with gastritis in proportion; variance analysis, age three group of diseases between F=1.51, P0.05, P=0.227, the difference was not statistically significant.
Detection and statistical analysis results: 2. genes of all tested strains were positive for cagA gene, EPIYA genotyping results: gastritis strains: EPIYA-ABD32 strain (86.5%), (2.7%), EPIYA-AD1 strain EPIYA-ABBD4 strain (10.8%); peptic ulcer (92%) strains: EPIYA-ABD23 strain, EPIYA-AB1 strain, EPIYA-ABBD1 strain ((4%) 4%); gastric cancer strain EPIYA-ABD19 strain (95%), EPIYA-ABBD1 (5%), strain analysis, EPIYA-ABD and non EPIYA-ABD group of type three disease card x 2=1.20, P0.05P=0.639. total strains, 74 strains of EPIYA-ABD, accounting for 90.2%, showed that the region, EPIYA type of Oriental type EPIYA-ABD, there was no significant difference between in each disease group.
VacA genotyping results: gastritis strains: s1c/i1/m1b13 strains (35.1%), s1c/i1/m224 (64.9% strains); peptic ulcer (4%) strains: s1a/i1/m1b1 strain, s1c/i1/m1b15 strain, s1c/i1/m29 strain (60%) (36%); gastric cancer strain s1c/i1/m1a1 strain s1c/i1/m1b9 strain (5%), (45%), s1c/i1/m210 strain (50%) not detected. Type S2, type I2 vacA.vacA m, M2 strains accounted for 52.4%, M1 accounted for 47.6%.logistic regression analysis, compared with peptic ulcer and gastritis between M1, OR=3.282,95% CI (1.138,9.468), P0.05P =0.028, the difference was statistically significant, showed that vacA M1 increased the patients at risk of developing peptic ulcer; type M2 strain ratio is higher than reported in the literature in East Asia and South Korea (8.65%), lower than the reported southeast areas of Vietnam (63.8%).
All tested strains were positive for oipA gene sequencing analysis, the switch state is "open" (all functional oipA), "CT" repeat feature "None" (6 strains), 1+4 (2 strains), 2+1 (2 strains), "2+1+1+1" (4 strain), 3+1 (56 strains), 3+2 (5 strains), 5+2 (1 strains), 6 (5 strains), 3 (1 strains) nine types.
The positive rate of dupA gene, 11 strains of chronic gastritis peptic ulcer (29.7%), 5 strains (20%) (of which 3 strains of gastric ulcer, duodenal ulcer, gastric cancer and 2 strains) 9 strains (45%). Duodenal ulcer group and duodenal ulcer group dupA gene between the chi square test, X ~2=0.76, P0.05P= 0.382, difference no statistical significance. DupA gene shows that as an important indicator of duodenal ulcer in this study, did not reflect this important role. Show that the marker gene dupA gene could not be used as a Hp in Fuzhou caused by duodenal ulcer.
Comprehensive analysis of EPIYA, vacA and dupA gene of EPIYA-ABD+dupA+s1/m1 strain in each disease group, 5.4% gastritis strains, 16% strains of peptic ulcer, gastric cancer accounted for 30% strains, chi square test, X ~2=6.340, P0.05P=0.036, vacA gene showed that EPIYA-ABD type, s1/m1 type, dupA positive strains also increased the risk of digestion ulcer and gastric cancer.
[Conclusion]
1. the cagA EPIYA typing of Hp strain in Fuzhou region embodies the characteristics of Hp strain in the eastern countries, with the main EPIYA-ABD type and no Western EPIYA-ABC strain.
2. vacA M1 type is a risk factor for peptic ulcer, and the proportion of vacA gene M2 is high. It is much higher than that in the M area, which is mainly M1B type, but lower than that in Southeast Asia.
3. it is still not considered that the dupA gene of strain Hp can be used as a marker gene for Hp induced duodenal ulcer disease in Fuzhou.
4.EPIYA-ABD, vacA gene s1/m1 and dupA three positive strains increased the risk of peptic ulcers and gastric cancer.

【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

【参考文献】