幽门螺杆菌cagA基因可变区多态性对其功能的影响

发布时间：2018-05-27 23:43

本文选题：幽门螺杆菌 + 细胞毒素相关蛋白A　；参考：《中国人民解放军军事医学科学院》2015年硕士论文

【摘要】：幽门螺杆菌(Helicobacter pylori,H.pylori)是一种革兰氏阴性微需氧菌,呈螺旋杆状[1,2]。H.pylori在全世界范围内感染率超过50%,在亚洲、非洲及南美洲等世界欠发达地区H.pylori感染率更高[3]。感染后可以长期定植于人类胃粘膜,是引起胃炎、消化性溃疡、胃粘膜相关的淋巴样组织淋巴瘤和胃腺癌的重要病原体[4-7]。1994年,H.pylori被世界卫生组织列为I类致癌因子,这也是目前为止唯一被列为I类致癌因子的病原细菌。H.pylori与胃十二指肠疾病关系密切,其细胞毒素相关蛋白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,通过特有的IV型分泌系统(type IV secretion system,TFSS)将Cag A蛋白注入宿主细胞,从而干扰宿主细胞功能,产生毒性作用[9]。本实验利用经过人工优化和人源化后的cag A不同亚型基因,构建成cag A六种不同亚型真核表达载体,分别转染胃上皮细胞AGS后,观察与分析比较了H.pylori Cag A EPIYA多态性对胃上皮细胞形态及IL-8表达的影响。与此同时,我们收集了河北地区131例H.pylori感染阳性的胃病患者标本,检测了感染H.pylori的Cag A EPIYA基序亚型,分析了其对胃病患者发生胃病种类及程度的影响。本实验分为如下三部分:一、cagA各亚型基因的优化、人工合成及其真核表达载体的构建通过NCBI Blast H.pylori cag A序列,共选取6条代表cag A各亚型基因的序列,分别为cag A 98-10、cag A Ca52、cag A NCTC11637、cag A F75、cag A PNGhigh85和cag A Shi470。上述基因利用密码子偏爱性、基因GC含量、二核苷酸序列等分析后,优化合成了4条全新的NCTC11637、F75、PNGhigh85和Shi470 cag AHS基因C端序列(cag A 98-10HS基因和cag A Ca52HS基因本实验室之前保存)。采用重组克隆试剂盒方法,将pcDNA3.1、cagA 98-10 N端分别与cagA NCTC11637HS、cag A SHI470HS、cag A PNGhigh85HS和cag A F75HS C端连接,构建了真核表达载体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,加上实验室原先构建的pc DNA3.1-cag A 98-10HS和pc DNA3.1-cag A Ca52HS,共获得cag A六种不同亚型的真核表达载体。二、不同亚型Cag A对AGS细胞形态及IL-8表达的影响对AGS细胞进行复苏、培养及传代,采用Western Blotting鉴定各型cagAHS真核表达载体瞬时转染入AGS后Cag A蛋白的表达。GAPDH为内参蛋白,空载体pc DNA3.1转染细胞为阴性对照,表明各型Cag A在AGS细胞内均成功表达,其在转染效率上的差异不具有统计学意义。各型cag AHS表达质粒瞬时转染AGS后于48h内观察细胞形态变化及IL-8表达,设空载体pc DNA3.1为阴性对照。转染后每隔6h进行细胞形态观察,于24h、36h对形成“蜂鸟状”细胞的数量进行统计分析,结果表明,各亚型Cag A均能引起发生“蜂鸟状”改变的细胞数量增加。较空载体对照,转染后24h和36h,Cag A六种亚型皆有非常显著差异(P0.001),Cag A ABCCC与其余五种Cag A亚型有非常显著差异(P0.001),Cag A ABDD与Cag A ABC和Cag A J-Western有非常显著差异(P0.001);24h时,Cag A ABDD与Cag A Amerindian有非常显著差异(P0.001);36h时,Cag A ABD分别与Cag A ABC和Cag A J-Western有非常显著差异(P0.01)。同时于转染后12h、36h对细胞分泌的IL-8进行检测,统计分析表明,转染后12h,较空载体对照,Cag A ABDD存在非常显著差异(P0.01);转染后36h,较空载体对照,Cag A ABD和Cag A J-Western存在非常显著差异(P0.01),Cag A ABDD和Cag A ABCCC存在非常显著差异(P0.001);Cag A Amerindian与Cag A ABD和Cag A J-Western分别存在非常显著差异(P0.01),与Cag A ABDD和Cag A ABCCC分别存在非常显著差异(P0.001);Cag A ABDD和Cag A ABCCC分别与Cag A ABD、Cag A ABC和Cag A J-Western存在非常显著差异(P0.001)。三、CagA EPIYA多态性与胃病的关系研究在中国,H.pylori感染及胃癌的发生均比较高,但有关H.pylori细胞毒素相关蛋白Cag A与胃病种类及程度的关系研究鲜有报道,而Cag A EPIYA基序与胃病及胃癌的关系研究的报道更少。本研究初步探究了H.pylori Cag A(+)EPIYA基序种类与胃病严重程度的关系。本研究从临床共收集到131例H.pylori感染阳性的胃病患者标本,其中经胃镜观察和病理诊断有37例属于慢性胃炎,42例胃溃疡,35例十二指肠溃疡及17例胃腺癌。经PCR扩增鉴定Cag A阳性117例,占总数89.3%。Cag A阳性中,有92例含有东亚型EPIYA-D基序,占78.6%,其中大多数为Cag A ABD(64.1%);有19例含有EPIYA-C基序,占总数的16.2%。Cag A ABD型在胃腺癌与非胃癌病例中分布具有统计学意义(P0.001),胃腺癌患者感染的幽门螺杆菌全部为Cag A ABD亚型,预测感染H.pylori为Cag A ABD亚型时患者患胃腺癌的风险性高。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2

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