本文選題：幽門螺桿菌 + 中性粒細(xì)胞激活蛋白�。� 參考：《第一軍醫(yī)大學(xué)》2007年博士論文

【摘要】： 幽門螺桿菌(Helicobacter pylori，Hp)是微需氧革蘭氏陰性螺桿菌。1983年，澳大利亞科學(xué)家J Robin Warren和Barry J Marshall首先發(fā)現(xiàn)了幽門螺桿菌，并且證實(shí)慢性胃炎和消化性潰瘍是由Hp感染所致，他們因?yàn)檫@一卓越的發(fā)現(xiàn)榮膺2005年諾貝爾生理學(xué)醫(yī)學(xué)獎(jiǎng)。幽門螺桿菌是世界范圍內(nèi)感染率最高的病原菌，全球人口感染率超過50％，它是慢性胃炎和消化性潰瘍的罪魁禍?zhǔn)�，并與胃腺癌、胃粘膜相關(guān)性淋巴樣組織淋巴瘤(gastric mucosal-associated lymphoid tissue lymphoma，MALT)的發(fā)生密切相關(guān)，1994年被世界衛(wèi)生組織確定為Ⅰ類致癌物。怎樣有效控制Hp感染，一直是國(guó)內(nèi)外學(xué)者關(guān)注的一大難題。 幽門螺桿菌的定植，伴隨著胃粘膜中性粒細(xì)胞、單核細(xì)胞和淋巴細(xì)胞的浸潤(rùn)，細(xì)菌誘導(dǎo)的免疫反應(yīng)不僅不能阻止細(xì)菌的定植，相反導(dǎo)致慢性胃部炎癥，粘膜損傷程度與中性粒細(xì)胞浸潤(rùn)程度密切相關(guān)。 幽門螺桿菌兩種主要的毒力因子是空泡毒素(vacuolating cytotoxin A，VacA)和中性粒細(xì)胞激活蛋白(HP neutrophil-activiting protein，HP-NAP)。中性粒細(xì)胞激活蛋白HP-NAP由napA基因編碼，分子量150kDa，是由4螺旋結(jié)構(gòu)單體構(gòu)成的十二聚體蛋白，對(duì)中性粒細(xì)胞、單核細(xì)胞有趨化作用，導(dǎo)致中性粒細(xì)胞浸潤(rùn)胃粘膜，并誘導(dǎo)中性粒細(xì)胞NADPH氧化酶激活，產(chǎn)生活性氧中間產(chǎn)物ROI(reactive oxygen intermediates，ROI)，引起粘膜炎癥和組織損傷。此外，HP-NAP能誘導(dǎo)中性白細(xì)胞和單核細(xì)胞表達(dá)β2整合素，以介導(dǎo)白細(xì)胞的粘附及吞噬作用，并介導(dǎo)免疫細(xì)胞間及免疫細(xì)胞與上皮細(xì)胞間的粘附作用；HP-NAP還能促進(jìn)單核細(xì)胞中組織因子合成和2型纖維蛋白溶酶原激活抑制分子的分泌；HP-NAP通過MAPK途徑激活中性粒細(xì)胞并激活肥大細(xì)胞脫顆粒和釋放前炎癥細(xì)胞因子IL-6；該蛋白中空可儲(chǔ)存鐵，其結(jié)構(gòu)類似大腸桿菌DNA結(jié)合蛋白Dps。最近研究發(fā)現(xiàn)，HP-NAP能促進(jìn)Th1免疫反應(yīng)，在感染位點(diǎn)形成以IL-12、IL6、IL8、TNF-a增高的Th1型細(xì)胞因子環(huán)境，影響感染的結(jié)局。HP-NAP抗原性強(qiáng)，大多數(shù)HP感染病人產(chǎn)生NAP抗體，用HP-NAP免疫小鼠能保護(hù)機(jī)體抵抗Hp的感染，保護(hù)率80％。表明HP-NAP可作為Hp多成分疫苗的候選抗原。 在HP-NAP的致病機(jī)理研究方面，由于它能激活中性粒細(xì)胞產(chǎn)生活性氧中間產(chǎn)物ROI，ROI能直接攻擊DNA而具致突變作用，提示ROI可能是致突變因子，與Hp相關(guān)性胃癌的發(fā)生有關(guān)。目前，人們對(duì)HP-NAP與Hp相關(guān)性胃癌之間的關(guān)系知之甚少，弄清它們之間的關(guān)系對(duì)Hp相關(guān)性胃癌的診斷和治療具有十分重要的意義。已知HP水溶性粗提物可上調(diào)中性粒細(xì)胞CXC趨化因子IL-8基因和生長(zhǎng)相關(guān)癌基因GROa的mRNA和蛋白表達(dá)，IL-8是致炎癥因子，GROa促進(jìn)新生血管生成，與癌癥的產(chǎn)生有關(guān)。NAP是否通過上調(diào)胃上皮細(xì)胞IL-8和GROa的表達(dá)發(fā)揮其致炎癥和致腫瘤作用，目前尚不清楚。 在Hp感染的治療方面，目前國(guó)內(nèi)外常用三聯(lián)藥物(如鉍劑+甲硝唑+抗生素)治療慢性胃炎和消化性潰瘍。但是，Hp容易產(chǎn)生耐藥性，導(dǎo)致抗生素治療失效。如果Hp未能根除，多達(dá)75-80％的患者很快又舊病復(fù)發(fā)。有學(xué)者認(rèn)為，病人口腔中存在Hp，可源源不斷地向胃內(nèi)供應(yīng)病菌，是導(dǎo)致感染復(fù)發(fā)的另一重要原因。此外，抗生素可引起菌群失調(diào)等毒副作用。鑒于抗生素療法不甚理想，疫苗被認(rèn)為是控制Hp感染最有效的方法。目前Hp苗研究的熱點(diǎn)主要集中在尿素酶(Ure)、毒力因子CagA、VacA和熱休克蛋白60(HSP60)等亞單位保護(hù)性抗原上。但尿素酶難以誘導(dǎo)出全面而穩(wěn)定的保護(hù)性免疫應(yīng)答；CagA和VacA變異較大；HSP60和人體自身蛋白有比較高的同源性，用HSP60免疫小鼠只能獲得局部保護(hù)性免疫應(yīng)答，并且會(huì)導(dǎo)致小鼠腸胃炎，其作為疫苗的安全性和有效性尚需進(jìn)一步驗(yàn)證。因此，迫切需要尋找新的合適的Hp疫苗候選抗原，深入探討其免疫機(jī)理，為Hp疫苗的發(fā)展提供理論基礎(chǔ)。多項(xiàng)實(shí)驗(yàn)證明，HP-NAP是疫苗研究的重要候選抗原，在HP-NAP抗原表位的研究方面，國(guó)內(nèi)外尚未見報(bào)道。 在本項(xiàng)研究中，我們首先構(gòu)建了原核表達(dá)系統(tǒng)，體外克隆和表達(dá)了幽門螺桿菌中性粒細(xì)胞激活蛋白HP-NAP。在此基礎(chǔ)上，應(yīng)用生物信息學(xué)、分子生物學(xué)、免疫學(xué)和生物化學(xué)等方法和技術(shù)著重從以下三個(gè)方面對(duì)HP-NAP的致病和免疫機(jī)理進(jìn)行了初步的探討。 一、幽門螺桿菌NAP抗體水平與胃癌的相關(guān)性及致病機(jī)理初探 應(yīng)用PCR技術(shù)檢測(cè)了Hp臨床菌株中napA基因存在狀況；應(yīng)用重組的NAP蛋白，采用ELISA方法檢測(cè)82例胃癌患者血清中NAP抗體含量，并與慢性胃炎和消化性潰瘍、正常人群血清中NAP抗體含量進(jìn)行比較，同時(shí)分析NAP抗體產(chǎn)生與患者年齡的關(guān)系。本研究還采用不同濃度的HP-NAP刺激SGC7901胃上皮細(xì)胞，測(cè)定細(xì)胞上清液中IL-8和癌細(xì)胞生長(zhǎng)因子GRO-α濃度的變化，對(duì)HP-NAP與胃上皮細(xì)胞的相互作用進(jìn)行了初步探討。 實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)：所有Hp臨床菌株中都存在napA基因，正常人血清中NAP抗體陽(yáng)性率為42.6％，而HPIgG抗體的陽(yáng)性率為55％，與我國(guó)人群中Hp感染率相似。HP-NAP抗體的陽(yáng)性率略低于HPIgG抗體的結(jié)果表明，雖然所有Hp臨床菌株均存在napA基因，但不同菌株NAP蛋白的表達(dá)有差異，與文獻(xiàn)報(bào)道一致。胃癌患者血清HP-NAP抗體陽(yáng)性率為97.5％，高于胃潰瘍92.9％和慢性胃炎患者85.7％的抗體陽(yáng)性率；胃癌患者NAP抗體水平顯著性高于胃炎患者，但與胃潰瘍患者無差異，表明NAP蛋白與胃癌的發(fā)生有一定相關(guān)性，NAP可能是胃癌發(fā)生的危險(xiǎn)因子。NAP抗體水平與患者年齡無明顯相關(guān)性。而且，NAP僅有輕微的刺激SGC7901胃上皮細(xì)胞株產(chǎn)生IL-8的作用，不能刺激SGC7901細(xì)胞產(chǎn)生GRO-α，說明NAP對(duì)胃上皮細(xì)胞的直接刺激作用十分有限，，它主要通過激活中性粒細(xì)胞發(fā)揮其致炎癥或致腫瘤的作用。 二、HP-NAP單克隆抗體的制備、鑒定及臨床意義 以天然的Hp全菌蛋白為抗原制備單克隆抗體，并用原核表達(dá)的重組HP-NAP蛋白鑒定篩選針對(duì)HP-NAP的抗體，對(duì)獲得的3株抗體進(jìn)行亞類鑒定、效價(jià)、特異性、臨床應(yīng)用等鑒定，結(jié)果獲得3株抗NAP蛋白的單抗，在Westernblot實(shí)驗(yàn)中，能與相應(yīng)的重組蛋白發(fā)生反應(yīng)�？筃AP單抗與其它腸道桿菌無交叉反應(yīng)。免疫組化鑒定發(fā)現(xiàn)，3株NAP單抗能與Hp臨床菌株發(fā)生反應(yīng)；E019單抗可以與Hp感染胃癌患者胃粘膜上的Hp特異性結(jié)合。 在單抗鑒定過程中，我們建立了用抗HP-NAP單克隆抗體檢測(cè)胃活檢標(biāo)本的免疫組化方法，可以用來比較胃炎、胃潰瘍和胃癌患者NAP蛋白的陽(yáng)性率，進(jìn)一步探討NAP蛋白與胃癌的相關(guān)性。在免疫組化實(shí)驗(yàn)中，我們發(fā)現(xiàn)HP-NAP單克隆抗體E019與HP感染胃癌組織發(fā)生強(qiáng)陽(yáng)性反應(yīng)，但與胃炎組織的反應(yīng)較弱，不顯色或微弱顯色，尚需加大例數(shù)作進(jìn)一步探討。而且，通過制備NAP單抗，可以建立更加特異和簡(jiǎn)便的診斷方法，用于Hp感染的診斷和預(yù)后的判斷。還可應(yīng)用鑒定出的NAP單抗作為靶分子，利用噬菌體肽庫(kù)技術(shù)篩選NAP具有免疫原性的抗原表位，用于Hp多表位疫苗的研究。 三、NAP生物信息學(xué)分析和B細(xì)胞抗原表位作圖 首先，應(yīng)用生物信息學(xué)技術(shù)，對(duì)HP-NAP的三維結(jié)構(gòu)和napA基因的ORF進(jìn)行了分析，由Linux操作系統(tǒng)預(yù)測(cè)的三維結(jié)構(gòu)顯示有多個(gè)a螺旋，提示HP-NAP中存在跨膜結(jié)構(gòu)，該蛋白是外膜蛋白；DNAMAN對(duì)napA基因的ORF分析顯示，它有兩個(gè)正向ORF和一個(gè)反向ORF，表明napA有許多不同的轉(zhuǎn)錄產(chǎn)物。接下來，依據(jù)HP-NAP的氨基酸序列，用DNAstar和EMBOSS在二級(jí)結(jié)構(gòu)預(yù)測(cè)、親水性和抗原性指數(shù)分析的基礎(chǔ)上，進(jìn)行B細(xì)胞抗原表位預(yù)測(cè)，發(fā)現(xiàn)有4個(gè)高抗原性肽段位于4-24，55-77，95-103和118-140氨基酸處，平均抗原性指數(shù)為1.0236，位于55-77氨基酸抗原性指數(shù)高達(dá)1.15并含有一個(gè)β轉(zhuǎn)角。然后，用抗HP-NAP單克隆抗體E019作為靶分子，對(duì)噬菌體隨機(jī)7肽庫(kù)分別進(jìn)行3輪“吸附-洗脫-擴(kuò)增”的篩選，對(duì)獲得的陽(yáng)性噬菌體克隆進(jìn)行篩選和鑒定，從篩選得到的陽(yáng)性克隆菌株提取單鏈DNA，進(jìn)行測(cè)序和分析，獲得模擬的B細(xì)胞表位，結(jié)果獲得3個(gè)模擬的B細(xì)胞表位XVXFXKV，LXHXPXX和XQKSHTV，分別位于14-20，60-66及131-137氨基酸處。而且，肽庫(kù)篩選獲得的抗原表位分別位于預(yù)測(cè)的4-24，55-77，118-140氨基酸處的高抗原性肽段中。結(jié)果說明生物信息學(xué)是蛋白質(zhì)抗原分析的良好工具，抗原表位能通過噬菌體展示肽庫(kù)篩選而獲得。本研究為Hp感染的免疫蛋白組學(xué)研究及疫苗研究提供了重要的信息。
[Abstract]:Helicobacter pylori ( Hp ) is Gram - negative Helicobacter pylori . In 1983 , Australian scientist J Robin Warren and Barry J Marshall first discovered H . pylori and confirmed that chronic gastritis and peptic ulcer were caused by Hp infection .






The colonization of Helicobacter pylori is associated with the infiltration of neutrophils , monocytes and lymphocytes in the gastric mucosa . The immune response induced by bacteria can not only prevent the colonization of bacteria , but also lead to chronic gastric inflammation , and the degree of mucosal injury is closely related to the degree of neutrophil infiltration .






Two major virulence factors of Helicobacter pylori are vacuolating cytotoxin A , VacA and HP neutrophil - activiting protein ( HP - NAP ) . The neutrophil activation protein HP - NAP is encoded by napa gene and has a molecular weight of 150 kDa . It is a decmer protein composed of 4 helix structural monomers . It can induce neutrophils and monocytes to infiltrate the gastric mucosa and induce the activation of the neutral granulocyte NADPH oxidase , which leads to the activation of the neutral granulocyte NADPH oxidase , which leads to inflammation and tissue damage of the mucous membrane .
HP - NAP can also promote the synthesis of tissue factor in monocytes and the activation of 2 - type plasminogen activator to inhibit the secretion of molecules .
HP - NAP activates neutrophils through MAPK pathways and activates mast cell degranulation and release pro - inflammatory cytokines IL - 6 ;
It is found that HP - NAP can promote Th1 immune response , form Th1 - type cytokine environment with increased IL - 12 , IL - 6 , IL8 and TNF - a in infection site , and affect the outcome of infection . HP - NAP has strong antigenicity . Most HP - infected patients produce NAP antibody . HP - NAP - immunized mice can protect organism against Hp infection , and the protective rate is 80 % . It is suggested that HP - NAP can be used as candidate antigen for Hp multi - component vaccine .






It is very important to study the relationship between HP - NAP and Hp - related gastric cancer . It is known that HP water - soluble crude extract can regulate the expression of IL - 8 gene and growth - related oncogene GROa in Hp - associated gastric cancer .






In the treatment of Hp infection , three drugs ( such as bismuth + metronidazole + antibiotics ) are commonly used in the treatment of chronic gastritis and peptic ulcer . However , Hp is easy to produce drug resistance , which leads to the failure of antibiotic treatment .
There was a large variation in the A and VacA .
HSP60 and human self - protein have high homology , but only partial protective immune responses can be obtained in mice immunized with HSP60 , and can lead to gastroenteritis in mice . As a result , there is an urgent need to find new candidate antigens for Hp vaccine . Therefore , it is urgent to find new candidate antigens for Hp vaccine and provide a theoretical basis for the development of Hp vaccine .






In this study , we constructed a prokaryotic expression system , cloned and expressed HP - NAP of Helicobacter pylori in vitro . On this basis , we discussed the pathogenic and immune mechanism of HP - NAP from the following three aspects : bioinformatics , molecular biology , immunology and biochemistry .






A preliminary study on the relationship between Helicobacter pylori NAP antibody level and gastric cancer and pathogenesis of gastric cancer






The presence of napa gene in Hp clinical isolates was detected by PCR technique .
The levels of NAP antibody in serum of 82 patients with gastric cancer were detected by ELISA and compared with chronic gastritis and peptic ulcer . The relationship between NAP antibody production and patient ' s age was analyzed .






The positive rate of HP - NAP antibody in gastric cancer patients was 97.5 % , which was higher than that in gastric ulcer 92.9 % and chronic gastritis 85.7 % .
NAP antibody levels in gastric cancer patients were significantly higher than those in patients with gastritis , but there was no difference between NAP and gastric cancer . NAP may be a risk factor for gastric cancer .






Preparation , Identification and Clinical Significance of Monoclonal Antibodies Against HP - NAP






The monoclonal antibody was prepared by using the natural Hp whole - bacterial protein as antigen , and the recombinant HP - NAP protein expressed by prokaryotic expression was used to identify the antibody against HP - NAP . The monoclonal antibody against the HP - NAP was identified by the recombinant HP - NAP protein . The results showed that 3 strains of anti - NAP monoclonal antibody could react with the corresponding recombinant protein .
E019 monoclonal antibody can specifically bind to Hp in gastric mucosa of patients with Hp infection .






In this paper , we have established an immunohistochemical method for the detection of gastric biopsy specimens with anti - HP - NAP monoclonal antibody , which can be used to compare the positive rate of NAP protein in gastritis , gastric ulcer and gastric cancer , and further study the correlation between NAP protein and gastric cancer .






III . NAP Bioinformatic Analysis and B - Cell Epitope Mapping






Firstly , the three - dimensional structure of HP - NAP and the ORF of napa gene were analyzed by bioinformatics . The three - dimensional structure predicted by the Linux operating system showed a number of a helix , suggesting that there is a transmembrane structure in HP - NAP , which is the outer membrane protein .
The results showed that four highly antigenic peptide fragments were located at amino acids of 4 - 24 , 55 - 77 , 95 - 103 and 118 - 140 . The results showed that four highly antigenic peptide fragments were located at amino acids of 4 - 24 , 55 - 77 , 95 - 103 and 118 - 140 respectively .
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R378

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