本文關(guān)鍵詞：不同消化性疾病來源的幽門螺桿菌毒力基因cagA、vacA、oipA、dupA的檢測及其意義　出處：《福建醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 幽門螺桿菌 cagA vacA oipA dupA 基因型 消化性疾病

【摘要】：[背景和目的] 幽門螺桿菌（Helicobacter pylori，以下簡稱Hp）是引起胃炎、消化性潰瘍、胃粘膜淋巴組織相關(guān)性淋巴瘤和胃癌的重要病原體。全球約有一半以上人口感染Hp，但在感染人群中只有約20%發(fā)生消化性疾病，且疾病的嚴(yán)重程度不同，其原因尚未闡明，可能與細(xì)菌基因高度多態(tài)性、宿主和環(huán)境等因素有關(guān)。Hp cagA、vacA、oipA、dupA四種毒力基因是幽門螺桿菌致病的重要因素。幽門螺桿菌感染的特點(diǎn)，主要體現(xiàn)在菌株基因多態(tài)性和地區(qū)分布差異性，且各種基因與疾病之間的關(guān)系也有地區(qū)差異性。本研究旨在調(diào)查胃癌高發(fā)的福州地區(qū)的Hp cagA及其C端的EPIYA基序、vacA和dupA分型以及oipA基因開關(guān)狀態(tài)，了解本地區(qū)的幽門螺桿菌基因多態(tài)性，并探討本地區(qū)消化性疾病與這些基因、性別和年齡之間的關(guān)系，以便對幽門螺桿菌的致病機(jī)制有更加深入的認(rèn)識。 [材料和方法] 1．幽門螺桿菌的分離與鑒定：從福建省腫瘤醫(yī)院，福建醫(yī)科大學(xué)附屬協(xié)和醫(yī)院和福建省立醫(yī)院采集的胃組織標(biāo)本（來自慢性胃炎、消化性潰瘍和胃癌患者），分離培養(yǎng)Hp，組織研磨后接種于布氏平板，于厭氧培養(yǎng)箱培養(yǎng)3-5d，經(jīng)尿素酶、過氧化氫酶及形態(tài)學(xué)鑒定確認(rèn)。 2．用高純度基因組DNA模板提取試劑盒（購自羅氏公司）提取Hp基因組DNA，并以其為模板，，PCR擴(kuò)增cagA3′端序列、vacA基因s區(qū)、i區(qū)、m區(qū)、oipA信號區(qū)、dupA（jhp917和jhp918）基因。 3． cagA3′端和oipA信號區(qū)PCR后送生工生物（上海）有限公司，sanger法，ABI-PRISM3730測序儀進(jìn)行基因測序，Bioedit分析軟件分析EPIYA基序類型和oipA基因開關(guān)狀態(tài)。 4．統(tǒng)計(jì)分析：運(yùn)用方差分析比較各疾病組之間年齡的差異，卡方檢驗(yàn)分析疾病與各基因之間的關(guān)系，logistic回歸分析各疾病組之間的基因差異。 [結(jié)果] 1.分離培養(yǎng)獲得82株幽門螺桿菌，其中分離自慢性胃炎患者37株，消化性潰瘍患者25株（其中胃潰瘍12株，十二指腸潰瘍13株），胃癌患者20株。疾病與性別和年齡的關(guān)系：單因素卡方分析胃炎，消化性潰瘍和胃癌組間性別差異，χ~2=7.39，P0.05，P=0.022，差異有統(tǒng)計(jì)學(xué)意義，表明消化性潰瘍和胃癌患者中男性比例高于胃炎患者中男性的比例；方差分析三組疾病間年齡差異，F(xiàn)=1.51，P0.05，P=0.227，差異無統(tǒng)計(jì)學(xué)意義。 2.基因檢測及統(tǒng)計(jì)學(xué)分析結(jié)果：所有被測菌cagA基因均為陽性， EPIYA分型結(jié)果：胃炎菌株：EPIYA-ABD32株（86.5%），EPIYA-AD1株（2.7%），EPIYA-ABBD4株（10.8%）；消化性潰瘍菌株： 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型三組疾病間卡方分析，χ2=1.20，P0.05P=0.639�？傮w菌株，EPIYA-ABD型74株，占90.2%，表明本地區(qū)，EPIYA分型以東方型的EPIYA-ABD為主，各疾病組之間無顯著差異。 vacA基因分型結(jié)果：胃炎菌株：s1c/i1/m1b13株(35.1%)，s1c/i1/m224株（64.9%）；消化性潰瘍菌株：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%）。未檢出s2型、i2型vacA。vacA m區(qū)，m2型菌株所占比例為52.4%，m1型所占比例為47.6%。logistic回歸分析，消化性潰瘍組與胃炎組間m1型相比，OR=3.282，95%可信區(qū)間為（1.138，9.468），P0.05P=0.028，差異有統(tǒng)計(jì)學(xué)意義，表明vacA m1型增加了患者發(fā)展為消化性潰瘍的危險(xiǎn)性；m2型菌株比例高于文獻(xiàn)報(bào)道的東亞地區(qū)的韓國（8.65%），低于文獻(xiàn)報(bào)道的東南亞地區(qū)的越南（63.8%）。 所有被測菌oipA基因均為陽性，測序分析其開關(guān)狀態(tài)均為“開”（均為功能性oipA），“CT”重復(fù)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）九種類型。 dupA基因陽性率，慢性胃炎為11株（29.7%），消化性潰瘍5株（20%）（其中胃潰瘍3株，十二指腸潰瘍2株），胃癌9株（45%）。十二指腸潰瘍組與非十二指腸潰瘍組間dupA基因卡方檢驗(yàn)，χ~2=0.76，P0.05P=0.382，差異無統(tǒng)計(jì)學(xué)意義。表明作為十二指腸潰瘍重要指標(biāo)的DupA基因在本研究中，未體現(xiàn)這一重要作用。表明dupA基因尚不能作為福州地區(qū)Hp致十二指腸潰瘍的標(biāo)記基因。 綜合分析EPIYA、vacA和dupA基因，EPIYA-ABD+dupA+s1/m1菌株在各疾病組間,胃炎菌株中占5.4%，消化性潰瘍菌株中占16%，胃癌菌株中占30%，卡方檢驗(yàn)χ~2=6.340，P0.05P=0.036，表明EPIYA-ABD型，vacA基因s1/m1型，dupA同時(shí)陽性菌株增加了患消化性潰瘍和胃癌的危險(xiǎn)性。 [結(jié)論] 1.福州地區(qū)Hp菌株的cagA EPIYA分型體現(xiàn)了東方國家Hp菌株的特征，以EPIYA-ABD型為主，未見西方型EPIYA-ABC菌株。 2. vacA m1型是消化性潰瘍的危險(xiǎn)因素，vacA基因m2型所占比例高。遠(yuǎn)比在m區(qū)中以m1b型為主的韓國要高，但比東南亞的越南的要低。 3.尚不能認(rèn)為Hp菌株dupA基因可以作為福州地區(qū)Hp致十二指腸潰瘍疾病的標(biāo)記基因。 4.EPIYA-ABD型、vacA基因s1/m1型和dupA三者同時(shí)陽性的菌株增加了患消化性潰瘍和胃癌的危險(xiǎn)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.

【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 黃贊松,唐國都,王超,李素艷,姜海行;廣西人上消化道疾病患者幽門螺桿菌cagA基因檢測及其臨床意義[J];世界華人消化雜志;2004年08期

2 尹焱,孫兆軍,張建中;北京人群感染幽門螺桿菌cagA分布特征[J];中華微生物學(xué)和免疫學(xué)雜志;2000年05期

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