發(fā)布時(shí)間：2018-05-27 23:43

  本文選題：幽門(mén)螺桿菌 + 細(xì)胞毒素相關(guān)蛋白A��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2015年碩士論文

【摘要】：幽門(mén)螺桿菌(Helicobacter pylori,H.pylori)是一種革蘭氏陰性微需氧菌,呈螺旋桿狀[1,2]。H.pylori在全世界范圍內(nèi)感染率超過(guò)50%,在亞洲、非洲及南美洲等世界欠發(fā)達(dá)地區(qū)H.pylori感染率更高[3]。感染后可以長(zhǎng)期定植于人類(lèi)胃粘膜,是引起胃炎、消化性潰瘍、胃粘膜相關(guān)的淋巴樣組織淋巴瘤和胃腺癌的重要病原體[4-7]。1994年,H.pylori被世界衛(wèi)生組織列為I類(lèi)致癌因子,這也是目前為止唯一被列為I類(lèi)致癌因子的病原細(xì)菌。H.pylori與胃十二指腸疾病關(guān)系密切,其細(xì)胞毒素相關(guān)蛋白A(cytotoxin associated gene A,Cag A)是H.pylori最重要的毒力因子之一[8]。H.pylori定植于胃上皮表面,通過(guò)特有的IV型分泌系統(tǒng)(type IV secretion system,TFSS)將Cag A蛋白注入宿主細(xì)胞,從而干擾宿主細(xì)胞功能,產(chǎn)生毒性作用[9]。本實(shí)驗(yàn)利用經(jīng)過(guò)人工優(yōu)化和人源化后的cag A不同亞型基因,構(gòu)建成cag A六種不同亞型真核表達(dá)載體,分別轉(zhuǎn)染胃上皮細(xì)胞AGS后,觀(guān)察與分析比較了H.pylori Cag A EPIYA多態(tài)性對(duì)胃上皮細(xì)胞形態(tài)及IL-8表達(dá)的影響。與此同時(shí),我們收集了河北地區(qū)131例H.pylori感染陽(yáng)性的胃病患者標(biāo)本,檢測(cè)了感染H.pylori的Cag A EPIYA基序亞型,分析了其對(duì)胃病患者發(fā)生胃病種類(lèi)及程度的影響。本實(shí)驗(yàn)分為如下三部分:一、cagA各亞型基因的優(yōu)化、人工合成及其真核表達(dá)載體的構(gòu)建通過(guò)NCBI Blast H.pylori cag A序列,共選取6條代表cag A各亞型基因的序列,分別為cag A 98-10、cag A Ca52、cag A NCTC11637、cag A F75、cag A PNGhigh85和cag A Shi470。上述基因利用密碼子偏愛(ài)性、基因GC含量、二核苷酸序列等分析后,優(yōu)化合成了4條全新的NCTC11637、F75、PNGhigh85和Shi470 cag AHS基因C端序列(cag A 98-10HS基因和cag A Ca52HS基因本實(shí)驗(yàn)室之前保存)。采用重組克隆試劑盒方法,將pcDNA3.1、cagA 98-10 N端分別與cagA NCTC11637HS、cag A SHI470HS、cag A PNGhigh85HS和cag A F75HS C端連接,構(gòu)建了真核表達(dá)載體pc DNA3.1-cag A NCTC11637HS,pc DNA3.1-cag A F75HS,pc DNA3.1-cag A PNGhigh85HS和pc DNA3.1-cag A Shi470HS,加上實(shí)驗(yàn)室原先構(gòu)建的pc DNA3.1-cag A 98-10HS和pc DNA3.1-cag A Ca52HS,共獲得cag A六種不同亞型的真核表達(dá)載體。二、不同亞型Cag A對(duì)AGS細(xì)胞形態(tài)及IL-8表達(dá)的影響對(duì)AGS細(xì)胞進(jìn)行復(fù)蘇、培養(yǎng)及傳代,采用Western Blotting鑒定各型cagAHS真核表達(dá)載體瞬時(shí)轉(zhuǎn)染入AGS后Cag A蛋白的表達(dá)。GAPDH為內(nèi)參蛋白,空載體pc DNA3.1轉(zhuǎn)染細(xì)胞為陰性對(duì)照,表明各型Cag A在A(yíng)GS細(xì)胞內(nèi)均成功表達(dá),其在轉(zhuǎn)染效率上的差異不具有統(tǒng)計(jì)學(xué)意義。各型cag AHS表達(dá)質(zhì)粒瞬時(shí)轉(zhuǎn)染AGS后于48h內(nèi)觀(guān)察細(xì)胞形態(tài)變化及IL-8表達(dá),設(shè)空載體pc DNA3.1為陰性對(duì)照。轉(zhuǎn)染后每隔6h進(jìn)行細(xì)胞形態(tài)觀(guān)察,于24h、36h對(duì)形成“蜂鳥(niǎo)狀”細(xì)胞的數(shù)量進(jìn)行統(tǒng)計(jì)分析,結(jié)果表明,各亞型Cag A均能引起發(fā)生“蜂鳥(niǎo)狀”改變的細(xì)胞數(shù)量增加。較空載體對(duì)照,轉(zhuǎn)染后24h和36h,Cag A六種亞型皆有非常顯著差異(P0.001),Cag A ABCCC與其余五種Cag A亞型有非常顯著差異(P0.001),Cag A ABDD與Cag A ABC和Cag A J-Western有非常顯著差異(P0.001);24h時(shí),Cag A ABDD與Cag A Amerindian有非常顯著差異(P0.001);36h時(shí),Cag A ABD分別與Cag A ABC和Cag A J-Western有非常顯著差異(P0.01)。同時(shí)于轉(zhuǎn)染后12h、36h對(duì)細(xì)胞分泌的IL-8進(jìn)行檢測(cè),統(tǒng)計(jì)分析表明,轉(zhuǎn)染后12h,較空載體對(duì)照,Cag A ABDD存在非常顯著差異(P0.01);轉(zhuǎn)染后36h,較空載體對(duì)照,Cag A ABD和Cag A J-Western存在非常顯著差異(P0.01),Cag A ABDD和Cag A ABCCC存在非常顯著差異(P0.001);Cag A Amerindian與Cag A ABD和Cag A J-Western分別存在非常顯著差異(P0.01),與Cag A ABDD和Cag A ABCCC分別存在非常顯著差異(P0.001);Cag A ABDD和Cag A ABCCC分別與Cag A ABD、Cag A ABC和Cag A J-Western存在非常顯著差異(P0.001)。三、CagA EPIYA多態(tài)性與胃病的關(guān)系研究在中國(guó),H.pylori感染及胃癌的發(fā)生均比較高,但有關(guān)H.pylori細(xì)胞毒素相關(guān)蛋白Cag A與胃病種類(lèi)及程度的關(guān)系研究鮮有報(bào)道,而Cag A EPIYA基序與胃病及胃癌的關(guān)系研究的報(bào)道更少。本研究初步探究了H.pylori Cag A(+)EPIYA基序種類(lèi)與胃病嚴(yán)重程度的關(guān)系。本研究從臨床共收集到131例H.pylori感染陽(yáng)性的胃病患者標(biāo)本,其中經(jīng)胃鏡觀(guān)察和病理診斷有37例屬于慢性胃炎,42例胃潰瘍,35例十二指腸潰瘍及17例胃腺癌。經(jīng)PCR擴(kuò)增鑒定Cag A陽(yáng)性117例,占總數(shù)89.3%。Cag A陽(yáng)性中,有92例含有東亞型EPIYA-D基序,占78.6%,其中大多數(shù)為Cag A ABD(64.1%);有19例含有EPIYA-C基序,占總數(shù)的16.2%。Cag A ABD型在胃腺癌與非胃癌病例中分布具有統(tǒng)計(jì)學(xué)意義(P0.001),胃腺癌患者感染的幽門(mén)螺桿菌全部為Cag A ABD亞型,預(yù)測(cè)感染H.pylori為Cag A ABD亞型時(shí)患者患胃腺癌的風(fēng)險(xiǎn)性高。
[Abstract]:Helicobacter pylori (H.pylori) is a gram-negative micro aerobic bacteria. The infection rate of spiral rod like [1,2].H.pylori is more than 50% in the world. In the less developed regions of Asia, Africa and South America, the H.pylori infection rate is higher than that of [3]. infection, and it can be planted in the human gastric mucosa for a long time. It is the cause of gastritis and digestion. Sexual ulcers, gastric mucosa associated lymphoid tissue lymphoma and important pathogens of gastric adenocarcinoma [4-7].1994 years, H.pylori is classified as the I carcinogen of the WHO, and this is so far the only pathogenic bacteria, which are listed as I carcinogens, closely related to gastroduodenal diseases, and its cytotoxin related protein A (cytotoxin). Associated gene A, Cag A) is one of the most important virulence factors of H.pylori, [8].H.pylori is planted on the surface of the gastric epithelium and injected into the host cell through a unique IV secretory system (type IV secretion system), which interferes with the host cell function and produces toxic effects. The different subtypes of CAG A were constructed into six different eukaryotic expression vectors of CAG A and transfected to AGS of gastric epithelial cells respectively. The effects of H.pylori Cag A EPIYA polymorphism on the morphology and IL-8 expression of gastric epithelial cells were compared and analyzed. At the same time, we collected 131 cases of gastropathy with H.pylori infection in Hebei. In this paper, we detected the Cag A EPIYA subtype of H.pylori infection and analyzed its effect on the type and degree of gastric disease in patients with gastric disease. The experiment was divided into three parts: 1, the optimization of each subtype of cagA, artificial synthesis and construction of the eukaryotic expression vector by NCBI Blast H.pylori CAG A sequence, and selected 6 subtypes representing CAG A. The sequences of the genes are CAG A 98-10, CAG A Ca52, CAG A NCTC11637, CAG A F75, and these genes are analyzed using codon bias, gene content and dinucleotide sequence. The A Ca52HS gene is preserved before the laboratory. Using the recombinant clone kit method, the pcDNA3.1 and the cagA 98-10 N ends are connected to the cagA NCTC11637HS, CAG A SHI470HS, CAG A. .1-cag A Shi470HS, coupled with the previously constructed PC DNA3.1-cag A 98-10HS and PC DNA3.1-cag A Ca52HS, obtained a total of six different subtypes of eukaryotic expression vectors. Two. The effects of different subtypes on the morphology and expression of the cells were resuscitated, cultured and passaged. After transient transfection, the expression of Cag A protein was expressed as internal reference protein and PC DNA3.1 transfected cells were negative control, indicating that all types of Cag A were expressed successfully in AGS cells, and the difference in transfection efficiency was not statistically significant. Each type CAG AHS expression plasmid was transiently transfected into AGS to observe cell morphologic changes after AGS DNA3.1. The expression of IL-8 and PC DNA3.1 were negative control. After transfection, the cell morphology was observed every 6h, and the number of "hummingbird" cells formed by 24h and 36h was statistically analyzed. The results showed that each subtype Cag A could cause the increase of the number of "hummingbird" changed cells. Compared with the empty carrier, 24h and 36h, Cag A six after transfection. There are very significant differences between the subtypes of the species (P0.001), and the difference between the Cag A ABCCC and the other five Cag A subtypes (P0.001). The Cag A ABDD and Cag A are very significant differences. There were very significant differences (P0.01). At the same time, after transfection, the IL-8 of the cells secreted by 12h and 36h was detected. The statistical analysis showed that after transfection, the Cag A ABDD had a very significant difference (P0.01) after the transfection, and the Cag A ABDD had a very significant difference (P0.01) after transfection, and there was a significant difference between the 36h and the empty carrier after transfection. There are very significant differences between Cag A Amerindian and Cag A ABD and Cag A J-Western respectively. There are very significant differences between the Cag A ABD and Cag A J-Western respectively. The relationship between A polymorphism and gastric disease is relatively high in China, H.pylori infection and gastric cancer are relatively high, but the relationship between the H.pylori cytotoxin related protein Cag A and the type and degree of gastric disease is rarely reported, but the relationship between the Cag A EPIYA motif and gastric disease and gastric cancer is less. This study preliminarily explored H.pylori Cag A (+) EP. The relationship between the type of IYA sequence and the severity of gastric disease. In this study, 131 cases of gastric disease with positive H.pylori infection were collected. Among them, 37 cases of chronic gastritis, 42 cases of gastric ulcers, 35 cases of duodenal ulcers and 17 cases of gastric adenocarcinoma were diagnosed by gastroscopy and pathological diagnosis. 117 cases of Cag A positive were identified by PCR amplification, accounting for 89.3%.Cag A. Of the positive, 92 cases contained the EPIYA-D motif of East Asia, accounting for 78.6%, most of which were Cag A ABD (64.1%), and 19 cases containing EPIYA-C motif, and the total number of 16.2%.Cag A ABD in the gastric adenocarcinoma and non gastric cancer cases was statistically significant (P0.001). All of the Helicobacter pylori infected by gastric adenocarcinoma were Cag A ABD subtype, predicting the infection. When ori is Cag A ABD subtype, the risk of gastric adenocarcinoma is high.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R735.2

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2 閆玉紅;肝腸鈣粘蛋白在胃腺癌中的表達(dá)及其病理關(guān)系的研究[D];河北醫(yī)科大學(xué);2015年

3 劉鹿;幽門(mén)螺桿菌cagA基因可變區(qū)多態(tài)性對(duì)其功能的影響[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2015年

4 杜天幸;Her-2、β-catenin、CIN 在胃腺癌中的表達(dá)及臨床意義[D];長(zhǎng)江大學(xué);2015年

5 謝振山;PTK7在胃腺癌中的表達(dá)及臨床意義[D];鄭州大學(xué);2015年

6 高德康;外周血循環(huán)微顆粒檢測(cè)方法的建立及其臨床應(yīng)用研究[D];蘇州大學(xué);2015年

7 魏紅;CD44v6 MMP-9 nm23在胃腺癌組織中的表達(dá)及意義[D];新疆醫(yī)科大學(xué);2007年

8 王名法;廣西地區(qū)人群DNA修復(fù)基因XPF基因RS#744154多態(tài)性與胃腺癌發(fā)病風(fēng)險(xiǎn)及臨床病理特征的關(guān)聯(lián)性研究[D];廣西醫(yī)科大學(xué);2011年

9 王錦洲;胃腺癌組織差異表達(dá)蛋白質(zhì)的分離與鑒定[D];福建醫(yī)科大學(xué);2013年

10 汪曉慶;胃腺癌血管生成擬態(tài)的初步研究[D];安徽醫(yī)科大學(xué);2010年

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