幽门螺杆菌中性粒细胞激活蛋白的致病与免疫机理探讨

发布时间：2018-07-09 17:48

本文选题：幽门螺杆菌 + 中性粒细胞激活蛋白　；参考：《第一军医大学》2007年博士论文

【摘要】： 幽门螺杆菌(Helicobacter pylori，Hp)是微需氧革兰氏阴性螺杆菌。1983年，澳大利亚科学家J Robin Warren和Barry J Marshall首先发现了幽门螺杆菌，并且证实慢性胃炎和消化性溃疡是由Hp感染所致，他们因为这一卓越的发现荣膺2005年诺贝尔生理学医学奖。幽门螺杆菌是世界范围内感染率最高的病原菌，全球人口感染率超过50％，它是慢性胃炎和消化性溃疡的罪魁祸首，并与胃腺癌、胃粘膜相关性淋巴样组织淋巴瘤(gastric mucosal-associated lymphoid tissue lymphoma，MALT)的发生密切相关，1994年被世界卫生组织确定为Ⅰ类致癌物。怎样有效控制Hp感染，一直是国内外学者关注的一大难题。幽门螺杆菌的定植，伴随着胃粘膜中性粒细胞、单核细胞和淋巴细胞的浸润，细菌诱导的免疫反应不仅不能阻止细菌的定植，相反导致慢性胃部炎症，粘膜损伤程度与中性粒细胞浸润程度密切相关。幽门螺杆菌两种主要的毒力因子是空泡毒素(vacuolating cytotoxin A，VacA)和中性粒细胞激活蛋白(HP neutrophil-activiting protein，HP-NAP)。中性粒细胞激活蛋白HP-NAP由napA基因编码，分子量150kDa，是由4螺旋结构单体构成的十二聚体蛋白，对中性粒细胞、单核细胞有趋化作用，导致中性粒细胞浸润胃粘膜，并诱导中性粒细胞NADPH氧化酶激活，产生活性氧中间产物ROI(reactive oxygen intermediates，ROI)，引起粘膜炎症和组织损伤。此外，HP-NAP能诱导中性白细胞和单核细胞表达β2整合素，以介导白细胞的粘附及吞噬作用，并介导免疫细胞间及免疫细胞与上皮细胞间的粘附作用；HP-NAP还能促进单核细胞中组织因子合成和2型纤维蛋白溶酶原激活抑制分子的分泌；HP-NAP通过MAPK途径激活中性粒细胞并激活肥大细胞脱颗粒和释放前炎症细胞因子IL-6；该蛋白中空可储存铁，其结构类似大肠杆菌DNA结合蛋白Dps。最近研究发现，HP-NAP能促进Th1免疫反应，在感染位点形成以IL-12、IL6、IL8、TNF-a增高的Th1型细胞因子环境，影响感染的结局。HP-NAP抗原性强，大多数HP感染病人产生NAP抗体，用HP-NAP免疫小鼠能保护机体抵抗Hp的感染，保护率80％。表明HP-NAP可作为Hp多成分疫苗的候选抗原。在HP-NAP的致病机理研究方面，由于它能激活中性粒细胞产生活性氧中间产物ROI，ROI能直接攻击DNA而具致突变作用，提示ROI可能是致突变因子，与Hp相关性胃癌的发生有关。目前，人们对HP-NAP与Hp相关性胃癌之间的关系知之甚少，弄清它们之间的关系对Hp相关性胃癌的诊断和治疗具有十分重要的意义。已知HP水溶性粗提物可上调中性粒细胞CXC趋化因子IL-8基因和生长相关癌基因GROa的mRNA和蛋白表达，IL-8是致炎症因子，GROa促进新生血管生成，与癌症的产生有关。NAP是否通过上调胃上皮细胞IL-8和GROa的表达发挥其致炎症和致肿瘤作用，目前尚不清楚。在Hp感染的治疗方面，目前国内外常用三联药物(如铋剂+甲硝唑+抗生素)治疗慢性胃炎和消化性溃疡。但是，Hp容易产生耐药性，导致抗生素治疗失效。如果Hp未能根除，多达75-80％的患者很快又旧病复发。有学者认为，病人口腔中存在Hp，可源源不断地向胃内供应病菌，是导致感染复发的另一重要原因。此外，抗生素可引起菌群失调等毒副作用。鉴于抗生素疗法不甚理想，疫苗被认为是控制Hp感染最有效的方法。目前Hp苗研究的热点主要集中在尿素酶(Ure)、毒力因子CagA、VacA和热休克蛋白60(HSP60)等亚单位保护性抗原上。但尿素酶难以诱导出全面而稳定的保护性免疫应答；CagA和VacA变异较大；HSP60和人体自身蛋白有比较高的同源性，用HSP60免疫小鼠只能获得局部保护性免疫应答，并且会导致小鼠肠胃炎，其作为疫苗的安全性和有效性尚需进一步验证。因此，迫切需要寻找新的合适的Hp疫苗候选抗原，深入探讨其免疫机理，为Hp疫苗的发展提供理论基础。多项实验证明，HP-NAP是疫苗研究的重要候选抗原，在HP-NAP抗原表位的研究方面，国内外尚未见报道。在本项研究中，我们首先构建了原核表达系统，体外克隆和表达了幽门螺杆菌中性粒细胞激活蛋白HP-NAP。在此基础上，应用生物信息学、分子生物学、免疫学和生物化学等方法和技术着重从以下三个方面对HP-NAP的致病和免疫机理进行了初步的探讨。一、幽门螺杆菌NAP抗体水平与胃癌的相关性及致病机理初探应用PCR技术检测了Hp临床菌株中napA基因存在状况；应用重组的NAP蛋白，采用ELISA方法检测82例胃癌患者血清中NAP抗体含量，并与慢性胃炎和消化性溃疡、正常人群血清中NAP抗体含量进行比较，同时分析NAP抗体产生与患者年龄的关系。本研究还采用不同浓度的HP-NAP刺激SGC7901胃上皮细胞，测定细胞上清液中IL-8和癌细胞生长因子GRO-α浓度的变化，对HP-NAP与胃上皮细胞的相互作用进行了初步探讨。实验结果发现：所有Hp临床菌株中都存在napA基因，正常人血清中NAP抗体阳性率为42.6％，而HPIgG抗体的阳性率为55％，与我国人群中Hp感染率相似。HP-NAP抗体的阳性率略低于HPIgG抗体的结果表明，虽然所有Hp临床菌株均存在napA基因，但不同菌株NAP蛋白的表达有差异，与文献报道一致。胃癌患者血清HP-NAP抗体阳性率为97.5％，高于胃溃疡92.9％和慢性胃炎患者85.7％的抗体阳性率；胃癌患者NAP抗体水平显著性高于胃炎患者，但与胃溃疡患者无差异，表明NAP蛋白与胃癌的发生有一定相关性，NAP可能是胃癌发生的危险因子。NAP抗体水平与患者年龄无明显相关性。而且，NAP仅有轻微的刺激SGC7901胃上皮细胞株产生IL-8的作用，不能刺激SGC7901细胞产生GRO-α，说明NAP对胃上皮细胞的直接刺激作用十分有限，，它主要通过激活中性粒细胞发挥其致炎症或致肿瘤的作用。二、HP-NAP单克隆抗体的制备、鉴定及临床意义以天然的Hp全菌蛋白为抗原制备单克隆抗体，并用原核表达的重组HP-NAP蛋白鉴定筛选针对HP-NAP的抗体，对获得的3株抗体进行亚类鉴定、效价、特异性、临床应用等鉴定，结果获得3株抗NAP蛋白的单抗，在Westernblot实验中，能与相应的重组蛋白发生反应。抗NAP单抗与其它肠道杆菌无交叉反应。免疫组化鉴定发现，3株NAP单抗能与Hp临床菌株发生反应；E019单抗可以与Hp感染胃癌患者胃粘膜上的Hp特异性结合。在单抗鉴定过程中，我们建立了用抗HP-NAP单克隆抗体检测胃活检标本的免疫组化方法，可以用来比较胃炎、胃溃疡和胃癌患者NAP蛋白的阳性率，进一步探讨NAP蛋白与胃癌的相关性。在免疫组化实验中，我们发现HP-NAP单克隆抗体E019与HP感染胃癌组织发生强阳性反应，但与胃炎组织的反应较弱，不显色或微弱显色，尚需加大例数作进一步探讨。而且，通过制备NAP单抗，可以建立更加特异和简便的诊断方法，用于Hp感染的诊断和预后的判断。还可应用鉴定出的NAP单抗作为靶分子，利用噬菌体肽库技术筛选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生物信息学分析和B细胞抗原表位作图首先，应用生物信息学技术，对HP-NAP的三维结构和napA基因的ORF进行了分析，由Linux操作系统预测的三维结构显示有多个a螺旋，提示HP-NAP中存在跨膜结构，该蛋白是外膜蛋白；DNAMAN对napA基因的ORF分析显示，它有两个正向ORF和一个反向ORF，表明napA有许多不同的转录产物。接下来，依据HP-NAP的氨基酸序列，用DNAstar和EMBOSS在二级结构预测、亲水性和抗原性指数分析的基础上，进行B细胞抗原表位预测，发现有4个高抗原性肽段位于4-24，55-77，95-103和118-140氨基酸处，平均抗原性指数为1.0236，位于55-77氨基酸抗原性指数高达1.15并含有一个β转角。然后，用抗HP-NAP单克隆抗体E019作为靶分子，对噬菌体随机7肽库分别进行3轮“吸附-洗脱-扩增”的筛选，对获得的阳性噬菌体克隆进行筛选和鉴定，从筛选得到的阳性克隆菌株提取单链DNA，进行测序和分析，获得模拟的B细胞表位，结果获得3个模拟的B细胞表位XVXFXKV，LXHXPXX和XQKSHTV，分别位于14-20，60-66及131-137氨基酸处。而且，肽库筛选获得的抗原表位分别位于预测的4-24，55-77，118-140氨基酸处的高抗原性肽段中。结果说明生物信息学是蛋白质抗原分析的良好工具，抗原表位能通过噬菌体展示肽库筛选而获得。本研究为Hp感染的免疫蛋白组学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 .
【学位授予单位】：第一军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R378

【参考文献】