【作者】 马一君；

【导师】 段广才；

【作者基本信息】 郑州大学 ， 流行病与卫生统计学， 2010， 博士

【摘要】 幽门螺杆菌(Helicobacter pylori,H.pylori)是一种专寄生于人胃黏膜的革兰阴性微需氧菌,世界上至少有50%人口感染H. pylori。许多研究表明,H. pylori感染后可导致胃炎和消化性溃疡等疾病,而且是胃癌、初级B淋巴瘤、硬化性胆管炎等疾病的重要因素。H. pylori的感染和发病涉及多个毒力因子。H.pylori接触胃粘膜上皮后可诱导表达一种毒力相关因子----粘膜接触诱导因子,是独立于cagA和vacA之外的一个重要毒力相关基因,由iceA (induced by contact with epithelium, iceA)基因编码,包括iceA1、iceA2两种等位基因。该基因可能通过调控相关毒素基因的表达等机制,参与Hp的致病作用。有研究发现,iceA基因与消化性溃疡及IL-8的黏膜浓度增高显著相关,而IL—8在H. pylori所致相关性炎症及疾病中发挥着重要作用。虽然国内外研究人员针对iceA基因与H. pylori所致临床疾病的相关性进行了大量研究,认为iceA基因(包括iceA1和iceA2基因)是慢性胃炎和十二指肠溃疡特异性相关基因,是一种与炎症和免疫损伤有关的毒力因子。但是iceA基因生物学功能及其致病机制仍不十分清楚。研究首先利用郑州市慢性萎缩性胃炎患者幽门螺杆菌临床分离株MEL-H.pylori 27,对iceA基因进行了克隆和序列分子特征分析；又根据同源重组原理,利用分子生物学方法进行了Hp27 iceA基因的插入失活突变,构建了iceA基因的Hp27突变株；然后进一步比较遗传背景相同的野生株和构建的Hp27 iceA基因突变株的重要特性,包括菌株的尿素酶活性、与细胞体外共培养时对胃粘膜上皮细胞的形态特征、细胞周期、增殖、凋亡等特性的影响；比较野生株和突变株对细胞的黏附性以及诱导细胞产生炎性细胞因子IL—8能力等炎症相关因素。为阐明iceA基因的功能及参与Hp致病的分子机制奠定了重要的实验基础。方法1幽门螺杆菌菌株的培养及基因组DNA的制备将临床分离株MEL-Hp27(Hp27)接种于布氏平板上,37℃微需氧条件下培养。3d后收获细菌,提取基因组DNA。2 Hp27 iceA基因的克隆2.1引物设计及PCR扩增参照GenBank公布的H.pylori菌株的iceA基因及其上下游核苷酸序列设计引物,用于扩增包括iceA基因在内的基因序列,扩增片段长度约790 bp。2.2 iceA基因克隆与测序PCR产物回收纯化后与pMD19-T载体连接并转化大肠杆菌DH5α,筛选含重组质粒的阳性克隆,测序并分析。2.3基因序列特征分析从GenBank上检索H.pylori菌株的iceA基因的同源基因序列,用Clustal W软件对检索到的基因序列进行多重序列比较,应用MEGA4.0等软件对基因及相应氨基酸序列进行同源比较,并构建系统发生树进行分析。3 iceA基因打靶载体的构建3.1将重组质粒pMD19-T-iceA及质粒pBluescript SKⅡ(一)分别酶切并连接,构建重组质粒pBS-iceA。3.2将重组质粒pBS-iceA和卡那霉素抗性基因扩增产物酶切并连接,构建出带卡那霉素抗性标记的突变打靶载体pBS-iceA-km。4 iceA基因突变株的构建与鉴定采用电穿孔方法将打靶载体pBS-iceA-km电转化入受体菌株野生型Hp27中,在普通布氏平板上培养48h,然后转涂于含卡那霉素布氏平板上继续培养3-5d,筛选出iceA基因突变株,经PCR及测序进行鉴定。5野生株和iceA基因突变株特性比较5.1采用尿素酶活性诊断试剂,检测和比较野生株Hp27和Hp27 iceA插入失活突变株的尿素酶活性差异。5.2将细菌与SGC7901细胞共同培养一定时间,观察和比较细胞形态学变化。5.3采用MTT比色法,检测野生株Hp27和Hp27 iceA插入失活突变株对SGC7901细胞增殖活性的影响并分析比较。5.4流式细胞仪检测野生株、突变株与SGC7901细胞共培养一定时间后细胞周期的改变并分析比较。5.5流式细胞仪检测野生株、突变株与SGC7901细胞共培养一定时间后细胞的凋亡率并分析比较。6 iceA基因的炎症相关因素研究6.1 Hp诱导SGC7901细胞分泌细胞因子IL-8的分析H. pylori27野生株与突变株分别与细胞共培养一定时间,诱导细胞分泌IL-8。收集培养上清液,用ELISA试剂盒定量检测IL-8的浓度并分析。6.2 H. pylori 27野生株与突变株对胃粘膜上皮细胞的黏附作用分析野生株与突变株分别与细胞共培养一定时间,用免疫学方法处理,在荧光倒置显微镜下观察,流式细胞仪检测并分析其粘附率。7统计学分析处理应用SPSS11.0软件分析各指标数据,计量资料以x±S表示,两组之间的比较采用t检验；多组之间的比较,根据资料特点分别采用单因素方差分析或重复测量的方差分析；两两比较采用最小显著差法(LSD),检验水准取a=0.05。结果1幽门螺杆菌分离株MEL-Hp27 iceA基因克隆及分析成功克隆幽门螺杆菌Hp27菌株iceA基因序列,扩增产物大小约为790 bp；重组质粒pMD19-T-iceA单酶切产生3820 bp片段,双酶切产生3000 bp和820 bp的2个片段；Hp27 iceA基因与大多数美国来源菌株同源性较高,均大于85%,与日本、印度等地区来源菌株同源性小于70%；Hp27 iceA基因一10区(TATA)位于起始密码子ATG上游的17bp处,起始密码子上游—7nt处有一个SD序列,起始密码子和上游cysE基因之间有两个8bp的串联可重复序列,其它地区分离菌株与Hp27 iceA基因特征有差异。2 MEL-Hp27 iceA基因突变株的构建采用基因重组的方法将卡那霉素抗性基因插入目的基因序列中,构建了突变打靶载体pBS-iceA-km;通过电穿孔转化方法将突变载体转化进入野生株,筛选出iceA基因突变株。用iceA基因两端序列设计引物分别扩增突变株和野生株的基因组DNA,结果突变株的扩增片段长度比野生株长约800bp,且突变株能扩增出卡那霉素抗性基因片段,野生株则不能。扩增产物的测序结果证明卡那霉素抗性基因插入目的基因序列中。3野生株和iceA基因突变株尿素酶活性比较将构建的突变株和野生株分别与尿素酶试剂作用,立即引起明显的颜色改变,野生株25min完全分解尿素酶试剂达到其最大吸光度值,且在此之前其吸光度值均高于突变株组。突变株组在50 min达到其最大吸光度值,且在作用25min后的吸光度值均高于野生株组,但两组之间的差异无统计学意义。4 iceA基因对SGC7901细胞生长的影响H.pylori突变株组和野生株组引起相似的细胞形态学改变,细胞拉伸,出现蜂鸟样改变；细菌和细胞共培养12h和18h,与对照组相比细胞活力轻度增加。共培养24h后,与对照组相比细胞活力则下降。在细菌与细胞共培养12h、18h、24h,野生株组和突变株组细胞增殖活性与空白组相比,差异无统计学意义(P>0.05);培养30h和36h各组细胞的增殖活性差异有统计学意义(P<0.05),其中,培养30h时突变株组的细胞增殖活性高于野生株组(P<0.05)；与对照组相比,野生株对细胞周期的影响主要表现为G2期的阻滞；而突变株对细胞周期的影响主要表现为S期的阻滞；野生株组和突变株组均可以诱导细胞产生明显凋亡,共培养24h及48h的凋亡率均高于对照组凋亡率(P<0.05),但野生株组和突变株组之间凋亡率的差异无统计学意义(P>0.05)。5 iceA基因的炎症相关因素研究将H.pylori分别与SGC7901细胞共培养8h和24h后,检测诱导IL-8的浓度,野生株组和突变株组与对照组相比,差异有统计学意义(P<0.05)。共培养8h野生株组和突变株组之间,差异无统计学意义(P>0.05),但共培养24h后,野生株组和突变株组间差异有统计学意义(P<0.05)；荧光显微镜下显示野生株和突变株均迅速粘附在SGC7901细胞表面,野生株和突变株的粘附率分别为32.49±2.96%和25.03±2.74%,但二组间差异无统计学意义(P>0.05)。结论1来自不同地区的幽门螺杆菌分离菌株iceA基因序列的分子特征有差异。2 Hp27菌株和Hp27 iceA基因突变株可以抑制SGC7901细胞增殖并诱导细胞凋亡；iceA基因的插入失活突变明显改变细胞增殖和细胞周期特征,对细胞凋亡率和粘附率的影响无统计学意义。3 Hp27菌株和Hp27 iceA基因突变株均可以诱导SGC7901细胞产生IL-8, iceA基因的插入失活突变导致细胞产生IL-8的浓度明显减少。更多还原

【Abstract】 Helicobacter pylori are Gram-negative spiral shaped bacteria that colonizes in human gastric mucosa. H. pylori infect more than 50% of humans globally. Now it is generally accepted that Helicobacter pylori cause gastric and duodenal ulcer diseases. Additionally, H. pylori also play an important role in the pathogenesis of gastric carcinoma, primary B-cell gastric lymphoma and Sclerosing Cholangitis. There are many virulence factors related with the diseases caused by H. pylori.The iceA genetic locus that is one of virulence factors independent on cagA or vacA was suggested being induced to express following contact with epithelial cells.Two allelic variants, iceA1 and iceA2, have been identified. The iceA gene is thought to be involved in the pathogenesis to induce the expression of genes related to virulence which is also associated with H.pylori infection. Some researchers reported that iceA has relationship with the increased content of IL-8 in gastric mucous membranes and ulcer diseases. And IL-8 plays an important role in H. pylori-related inflammation and diseases. Several intriguing epidemiological studies have been reported about the Clinical relevance of the allelic iceA status of Helicobacter pylori isolated from different regions. While iceA alleles, iceAl or iceA2, show some specific relationship for gastroduodenal pathologies and is a virulence factor associated with inflammation and injury immunoreaction. However the function and the pathogenic role of iceA in Helicobacter pylori still is unclear.In the present study, iceA gene of H.pyloir MEL-Hp27 isolated from Chinese patients with chronic atrophy gastritis was cloned and its molecular character was analyzed. Then, insertional mutagenesis was carried out within the gene of H. pylori 27 by the method of molecule biology according to homologous recombination and the mutant strain was constructed. After that, difference of activities of urease between wild strain Hp27 and iceA isogenic mutant strain were assessed. Then we compared the mutant with wild strain as regards their characters, including the state, cell cycle, proliferation and apoptosis of SGC7901 cells, through co-culture of the bacteria and gastric epithelial cells. Finally, the effect of iceA gene on adherence of the bacteria to gastric SGC cells and on Helicobacter pylori associated IL-8 production from the epithelial cells was also assessed. This study provided the important experimental evidences for elucidating the function and the pathogenic role of the iceA gene in Helicobacter pylori.Methods1 Cluture of H.pylori and extraction of the genomic DNAH.pylori MEL-Hp27(Hp27) isolated from the gastric mucosa of patient with chronic atrophic gastritis was grown microarobically at 37℃for 3d in brucella broth plates.The H.pylori cells were harvested and the genomic DNA was extracted.2 Clone of iceA gene of Hp272.1 Primers designing and PCRReferring to the sequence of H.pylori strains published on GENBANK, primers were designed according to the nucleotide acid sequences of iceA gene and the sequences located in the upstream or downstream of the iceA gene. Then Hp27 iceA gene was amplified and cloned by using a polymerase chain reaction based approach.2.2 Cloning and sequencing the iceA gene of MEL-Hp27The purified product of PCR was subcloned into pMD19-T vector and transformed into DH5a.The positive clone was identified and the sequence was confirmed by DNA sequencing.2.3 Analysis of characteristics of iceA geneThe homology of iceA gene of MEL-Hp27 strain was compared with other Hp strains isolated from different regions published on GENBANK in nucleotide acid and the phylogenetic tree was constructed.3 Construction of targeting vector of iceA3.1 Recombinant plasmid pMD19-T-iceA and pBluescript SKⅡ(-) were digested with enzyme and connected into recombinant plasmid pBS-iceA.3.2 Recombinant plasmid pBS-iceA and the amplified kanamycin resistance gene were digested and connected into targeting vector of iceA mutant with kanamycin resistance gene pBS-iceA-km.4 Construction and identification of iceA mutant strain The recipient strain Hp27 was electrotransformed with the targeting vector pBS-iceA-km. The kanamycin resistance transformants were screened from brucella broth plates and identified by PCR and sequencing.5 Compare the characteristics between the wild and iceA mutant strains5.1 The activities of urease of wild strain Hp27 and iceA isogenic mutant were assayed by the urease-testing reagents5.2 Morphological changes of cells were observed and analyzed after a period of time through co-culture of the bacteria and gastric epithelial cells.5.3 The cellular proliferation was examined and compared by methyl thiazolyl tetrazolium(MTT) method through co-culture of the bacteria and gastric epithelial cells.5.4 Cell cycle were determined and analyzed by flow cytometry through co-culture of the bacteria and gastric epithelial cells for a period of time.5.5 Apoptosis of SGC7901 cells were determined and analyzed by flow cytometry through co-culture of the bacteria and gastric epithelial cells for a period of time.6 Study of the factors related with inflammation about iceA gene6.1 Analysis of the concentration of IL-8 induced by Hp27 from the SGC7901 cellsIL-8 was induced from the cells through co-culture of the wild or mutant Hp and gastric epithelial cells for a period of time. The concentration of IL-8 in the culture supernatants was determined and analyzed by enzyme-linked immunosorbent assay (ELISA)6.2 Analysis of the adherence of the wild or mutant strains to gastric SGC cells.After co-cultured the wild or mutant Hp and gastric epithelial cells for a period of time, they were treated using method of immunology. The adherence of bacteria to the cells was observed by fluorescent microscopy and adhesion to the cells was examined by flow cytometry.7 Statistical analysisThe experimental data were analyzed by the software of SPSS 11.0.The results of numerical data were expressed with (?)±S. T test was used to compare the statistical difference between two groups. According to the features of data, one-way analysis of variance or repeated measures analysis of variance were applied to test the statistical difference among three or more groups. The significance level was set at a=0.05Results1 Cloning and analysis of gene iceA of Helicobacter pylori isolatedThe iceA gene of Hp27 strain was successfully cloned. The PCR product was about 790bp. The recombinant plasmid pMD 19-T-iceA was digested by one restriction enzyme to produce a 3 820bp DNA fragment and digested by two enzymes to produce 3 OOObp and 820bp DNA fragments. Sequence analysis showed that the iceA gene of MEL-Hp27 have more than 85% homology with H.pylori American strains and less than 70% of homology with H.pylori Japanese or Indian strains. The-10 box was located in the up-stream of iceA gene and the distance from ATG was 17bp. The SD sequence is located in the up-stream of iceA gene and the distance from ATG was 7bp.There are two avariable numbers of tandem repeats between ATG and the up-stream gene cysE. There are differences about the characters of the gene between Hp27 strains and other Helicobacter pylori strains from other regions.2 Construction of MEL-Hp27 iceA mutant strainIn this study, Kanamycin resistance gene was inserted into the target gene by the method of gene recombination to construct the targeting vector pBS-iceA-km, with which the recipient strain Hp27 was electrotransformed. IceA mutant strain was screened on brucella broth plates with kanamycin and identified.DNA fragments, which were amplified from genomic DNA of mutant or wild strains separately by primers designed according to the flanking sequences of the iceA gene, from mutant strains are longer 800bp than the wild. Kanamycin resistance gene can be amplified from the mutant strains but not wild. The sequencing result of amplified product also showed that Kanamycin resistance gene had been inserted into the target gene 3 Comparison the activities of urease of wild strains with iceA gene mutantsRemarkable color change can be seen immediately when wild strain and constructed isogenic mutant were assayed by the urease-testing reagents. After being treated for 25 minutes, the OD values of wild strains reached a climax when urease had been decomposed completely. Before that, all its OD values at different time point were higher than mutant strains. The OD values of mutant strains reached a climax until 50 minutes later. There are some differences between two groups but not significant.4 The effect of iceA gene on growth state of SGC7901Cell elongation and hummingbird morphology were showed through co-culture of the bacteria and gastric epithelial cells for 48h and similar changes of cell morphology were observed in wild and mutant strains groups. The MTT assay showed that the vigor of cells increased slightly after co-cultured for 12h or 18h with Hp compared with contral group, but began to descend after co-cultured for 24h. There was no remarkable difference in cellular proliferation among all groups when co-cultured for 12h,18h or 24h, but there were distinct differences when co-cultured for 30h or 36h (P<0.05).Especially cellular proliferation of mutant group was significantly higher than wild group when co-cultured for 30h (P<0.05).The primary influence of wild strain on the cell cycle was that it arrested the cell cycle of SGC at G2 phase compared with contral group. But for mutant group, it arrested at S phase. SGC7901 cells infected with wild or mutant strains,24h or 48h, all displayed a significant increase in apoptosis, with statistically significant levels of apoptosis compared to uninfected cells (P<0.05). However there were no significant differences (P>0.05) between mutant group and wild group.5 Study of the factors related with inflammation about iceA geneAfter SGC7901 cells were co-cultured with Helicobacter for 8h or 24h, the concentration of IL-8 in the culture supernatants was determined and analyzed. Significant differences were observed between SGC cells interacting with the Hp strains (wild and mutant) and the uninfected cells (P<0.05). There were significant differences for the concentration of IL-8 between wild and mutant groups if co-cultured for 24h (P<0.05), but not for 8h ((P>0.05).When SGC7901 cells were co-cultured with Helicobacter, both of wild and mutant strains were bounded to cells rapidly which was confirmed by fluorescent microscopy. Adhesion to cells was examined by flow cytometry. The mean numbers of wild or mutant strains adhering to cells were 32.49±2.96% and 25.03±2.74%, respectively. The difference was not statistically significant (P>0.05).Conclusions1 There are some differences for iceA gene among Helicobacter pylori strains from different regions.2 The mutant of iceA gene had significantly effect on the activities of proliferation and the cell cycle of SGC7901 cells. However, it did not change adhesion and apoptosis of Hp to cells significantly.3 IL-8 was induced by Hp27 from the SGC7901 cells. The mutant of iceA gene decreased the concentration of IL-8 distinctly.更多还原