本文選題：幽門螺桿菌 + 中性粒細胞激活蛋白��； 參考：《第一軍醫(yī)大學》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，澳大利亞科學家J Robin Warren和Barry J Marshall首先發(fā)現(xiàn)了幽門螺桿菌，并且證實慢性胃炎和消化性潰瘍是由Hp感染所致，他們因為這一卓越的發(fā)現(xiàn)榮膺2005年諾貝爾生理學醫(yī)學獎。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首，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，一直是國內(nèi)外學者關(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胞、單核細胞和淋巴細胞的浸潤，細菌誘導的免疫反應不僅不能阻止細菌的定植，相反導致慢性胃部炎癥，粘膜損傷程度與中性粒細胞浸潤程度密切相關(guān)。 幽門螺桿菌兩種主要的毒力因子是空泡毒素(vacuolating cytotoxin A，VacA)和中性粒細胞激活蛋白(HP neutrophil-activiting protein，HP-NAP)。中性粒細胞激活蛋白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，對中性粒細胞、單核細胞有趨化作用，導致中性粒細胞浸潤胃粘膜，并誘導中性粒細胞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能誘導中性白細胞和單核細胞表達β2整合素，以介導白細胞的粘附及吞噬作用，并介導免疫細胞間及免疫細胞與上皮細胞間的粘附作用；HP-NAP還能促進單核細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途徑激活中性粒細胞并激活肥大細胞脫顆粒和釋放前炎癥細胞因子IL-6；該蛋白中空可儲存鐵，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Th1免疫反應，在感染位點形成以IL-12、IL6、IL8、TNF-a增高的Th1型細胞因子環(huán)境，影響感染的結(jié)局。HP-NAP抗原性強，大多數(shù)HP感染病人產(chǎn)生NAP抗體，用HP-NAP免疫小鼠能保護機體抵抗Hp的感染，保護率80％。表明HP-NAP可作為Hp多成分疫苗的候選抗原。 在HP-NAP的致病機理研究方面，由于它能激活中性粒細胞產(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)中性粒細胞CXC趨化因子IL-8基因和生長相關(guān)癌基因GROa的mRNA和蛋白表達，IL-8是致炎癥因子，GROa促進新生血管生成，與癌癥的產(chǎn)生有關(guān)。NAP是否通過上調(diào)胃上皮細胞IL-8和GROa的表達發(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，導致抗生素治療失效。如果Hp未能根除，多達75-80％的患者很快又舊病復發(fā)。有學者認為，病人口腔中存在Hp，可源源不斷地向胃內(nèi)供應病菌，是導致感染復發(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，疫苗被認為是控制Hp感染最有效的方法。目前Hp苗研究的熱點主要集中在尿素酶(Ure)、毒力因子CagA、VacA和熱休克蛋白60(HSP60)等亞單位保護性抗原上。但尿素酶難以誘導出全面而穩(wěn)定的保護性免疫應答；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性免疫應答，并且會導致小鼠腸胃炎，其作為疫苗的安全性和有效性尚需進一步驗證。因此，迫切需要尋找新的合適的Hp疫苗候選抗原，深入探討其免疫機理，為Hp疫苗的發(fā)展提供理論基礎(chǔ)。多項實驗證明，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，國內(nèi)外尚未見報道。 在本項研究中，我們首先構(gòu)建了原核表達系統(tǒng)，體外克隆和表達了幽門螺桿菌中性粒細胞激活蛋白HP-NAP。在此基礎(chǔ)上，應用生物信息學、分子生物學、免疫學和生物化學等方法和技術(shù)著重從以下三個方面對HP-NAP的致病和免疫機理進行了初步的探討。 一、幽門螺桿菌NAP抗體水平與胃癌的相關(guān)性及致病機理初探 應用PCR技術(shù)檢測了Hp臨床菌株中napA基因存在狀況；應用重組的NAP蛋白，采用ELISA方法檢測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抗體含量進行比較，同時分析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胃上皮細胞，測定細胞上清液中IL-8和癌細胞生長因子GRO-α濃度的變化，對HP-NAP與胃上皮細胞的相互作用進行了初步探討。 實驗結(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抗體陽性率為42.6％，而HPIgG抗體的陽性率為55％，與我國人群中Hp感染率相似。HP-NAP抗體的陽性率略低于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蛋白的表達有差異，與文獻報道一致。胃癌患者血清HP-NAP抗體陽性率為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胃上皮細胞株產(chǎn)生IL-8的作用，不能刺激SGC7901細胞產(chǎn)生GRO-α，說明NAP對胃上皮細胞的直接刺激作用十分有限，，它主要通過激活中性粒細胞發(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，并用原核表達的重組HP-NAP蛋白鑒定篩選針對HP-NAP的抗體，對獲得的3株抗體進行亞類鑒定、效價、特異性、臨床應用等鑒定，結(jié)果獲得3株抗NAP蛋白的單抗，在Westernblot實驗中，能與相應的重組蛋白發(fā)生反應�？筃AP單抗與其它腸道桿菌無交叉反應。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；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單克隆抗體檢測胃活檢標本的免疫組化方法，可以用來比較胃炎、胃潰瘍和胃癌患者NAP蛋白的陽性率，進一步探討NAP蛋白與胃癌的相關(guān)性。在免疫組化實驗中，我們發(fā)現(xiàn)HP-NAP單克隆抗體E019與HP感染胃癌組織發(fā)生強陽性反應，但與胃炎組織的反應較弱，不顯色或微弱顯色，尚需加大例數(shù)作進一步探討。而且，通過制備NAP單抗，可以建立更加特異和簡便的診斷方法，用于Hp感染的診斷和預后的判斷。還可應用鑒定出的NAP單抗作為靶分子，利用噬菌體肽庫技術(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生物信息學分析和B細胞抗原表位作圖 首先，應用生物信息學技術(shù)，對HP-NAP的三維結(jié)構(gòu)和napA基因的ORF進行了分析，由Linux操作系統(tǒng)預測的三維結(jié)構(gòu)顯示有多個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napA基因的ORF分析顯示，它有兩個正向ORF和一個反向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在二級結(jié)構(gòu)預測、親水性和抗原性指數(shù)分析的基礎(chǔ)上，進行B細胞抗原表位預測，發(fā)現(xiàn)有4個高抗原性肽段位于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1.15并含有一個β轉(zhuǎn)角。然后，用抗HP-NAP單克隆抗體E019作為靶分子，對噬菌體隨機7肽庫分別進行3輪“吸附-洗脫-擴增”的篩選，對獲得的陽性噬菌體克隆進行篩選和鑒定，從篩選得到的陽性克隆菌株提取單鏈DNA，進行測序和分析，獲得模擬的B細胞表位，結(jié)果獲得3個模擬的B細胞表位XVXFXKV，LXHXPXX和XQKSHTV，分別位于14-20，60-66及131-137氨基酸處。而且，肽庫篩選獲得的抗原表位分別位于預測的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篩選而獲得。本研究為Hp感染的免疫蛋白組學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 .
【學位授予單位】：第一軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R378

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