本文選題：spoT基因 + 幽門螺桿菌��； 參考：《山東大學(xué)》2010年碩士論文

【摘要】： 目的 幽門螺桿菌(Helicobacter pylori, H pylori)是一個(gè)重要的人類病原菌,革蘭染色陰性、螺桿狀、微需氧菌,特異性寄生于胃粘膜,其感染可導(dǎo)致慢性胃炎、消化性潰瘍、胃粘膜相關(guān)淋巴組織(MALT)淋巴瘤及胃癌的發(fā)生,1994年世界衛(wèi)生組織國(guó)際癌癥研究機(jī)構(gòu)(IARC)已將其列為Ⅰ類致癌因子�，F(xiàn)在世界人口中大約一半被H pylori感染,中國(guó)是感染的高發(fā)區(qū),并且在不接受抗菌治療的情況下被感染者一般終生帶菌。盡管如此,迄今為止Hpylori的傳播途徑和持續(xù)性感染機(jī)制還不明確,由此制約了針對(duì)Hpylori感染的預(yù)防和治療。 H pylori在定植于宿主胃粘膜細(xì)胞的過程中可誘發(fā)強(qiáng)烈的炎癥反應(yīng),這一過程刺激宿主免疫細(xì)胞產(chǎn)生大量的活性氧分子(ROS)和活性氮中介物(RNI)。雖然受到大量ROS／RNI的氧化刺激,H pylori卻能夠成功地在胃粘膜定植并生存。這說(shuō)明H pylori具有有效的適應(yīng)氧應(yīng)激系統(tǒng)。研究發(fā)現(xiàn)H pylori具有較多抗氧應(yīng)激的效應(yīng)基因,但調(diào)控基因卻較少,因此H pylori抗氧應(yīng)激調(diào)控機(jī)制尚不明確。研究表明宿主通過細(xì)胞免疫防御幽門螺桿菌感染,特別是巨噬細(xì)胞的吞噬作用,然而幽門螺桿菌可以有效的應(yīng)對(duì)細(xì)胞免疫特別是Th1細(xì)胞免疫應(yīng)答,其中vacA與尿素酶基因在其免疫逃逸中具有重要作用。然而,幽門螺桿菌免疫逃逸調(diào)控機(jī)制卻研究甚少。 spoT基因是一個(gè)全局性的轉(zhuǎn)錄后調(diào)控基因,其功能最早發(fā)現(xiàn)是調(diào)控大腸桿菌的嚴(yán)謹(jǐn)應(yīng)答。所謂嚴(yán)謹(jǐn)應(yīng)答是指細(xì)菌為了應(yīng)對(duì)氨基酸饑餓而發(fā)生的基因表達(dá)模式的劇烈變化也稱作“營(yíng)養(yǎng)應(yīng)激應(yīng)答”�，F(xiàn)在將細(xì)菌在各種不利生存環(huán)境下發(fā)生的基因表達(dá)模式的改變統(tǒng)稱為“嚴(yán)謹(jǐn)應(yīng)答”。在H pylori基因組中也含有這個(gè)基因,最近研究發(fā)現(xiàn)在惡劣環(huán)境下,spoT基因也可以調(diào)控H pylori的嚴(yán)謹(jǐn)應(yīng)答,但機(jī)制未明。Hpylori在宿主體內(nèi)定植過程中受到的氧應(yīng)激與吞噬可以看做一種嚴(yán)謹(jǐn)環(huán)境,因此我們的研究主要探討spoT基因在H pylori在宿主體長(zhǎng)期定植適應(yīng)宿主免疫應(yīng)答過程中所起的作用,并對(duì)觀察到的現(xiàn)象作出初步解釋,為Hpylori的預(yù)防和治療提供線索。 方法 1、將已構(gòu)建好的H pylori spoT基因缺失突變株和野生株分別多次經(jīng)口感染蒙古沙鼠,分別于末次感染后的第2、4、6、8周脫頸處死蒙古沙鼠,取其胃粘膜組織進(jìn)行分離培養(yǎng)和革蘭染色初步鑒定細(xì)菌,應(yīng)用實(shí)時(shí)熒光定量PCR絕對(duì)定量方法對(duì)H pylori進(jìn)行鑒定和數(shù)量測(cè)定。 2、用人外周血中免疫細(xì)胞分別刺激H pylori spoT基因缺失突變株和野生株,并取兩個(gè)作用時(shí)間點(diǎn)：30分鐘和60分鐘。提取標(biāo)本RNA,反轉(zhuǎn)錄后應(yīng)用實(shí)時(shí)熒光定量PCR分別測(cè)定比較它們抗氧應(yīng)激相關(guān)基因(sodB、katA、tasA、rocF)、毒力基因(cagA、vacA、napA)、降解受損蛋白相關(guān)基因(clpP、clpX)在mRNA水平的表達(dá)情況。 3、動(dòng)物實(shí)驗(yàn)中處死蒙古沙鼠時(shí)同時(shí)摘眼球取血,收集每次每組血清保存于-80℃,酶聯(lián)免疫吸附測(cè)定方法測(cè)定并比較外周血液中的DNA氧化損傷標(biāo)志物8-羥基脫氧鳥苷(8-OHdG)的含量,以此反應(yīng)不同菌株在體內(nèi)產(chǎn)生活性氧的水平,間接反應(yīng)不同菌株感染誘導(dǎo)產(chǎn)生免疫細(xì)胞的水平。 4、用超氧化物陰離子熒光檢測(cè)探針DHE裝載人外周血的免疫細(xì)胞,Hpylori spoT基因缺失突變株和野生株分別作用于一定量的人外周血的免疫細(xì)胞不同時(shí)間后,用流式細(xì)胞術(shù)測(cè)定比較不同菌株在體外產(chǎn)生活性氧水平。 結(jié)果 1、動(dòng)物實(shí)驗(yàn)表明H pylori spoT基因缺失突變株和野生株均可在蒙古沙鼠胃內(nèi)成功定植；H pylori spoT基因缺失突變株在蒙古沙鼠胃粘膜的定植能力明顯低于野生株,在第8周時(shí)甚至幾乎檢測(cè)不到突變株定植。 2、spoT基因缺失突變株中大部分抗氧應(yīng)激基因(sodB、katA、tasA、rocF)、毒力基因(cagA、vacA、napA)在mRNA水平表達(dá)異常,兩個(gè)與降解受損蛋白相關(guān)的關(guān)鍵基因(clpP、clpX)表達(dá)降低。 3、蒙古沙鼠感染H pylori野生菌株組(W組)與感染突變菌株組(M組)比較,末次感染后第2、4、6周蒙古沙鼠外周血中8-OHdG含量?jī)山M相差不大,但第8周M組明顯少于W組。 4、在各個(gè)時(shí)間點(diǎn)進(jìn)行比較發(fā)現(xiàn)：H pylori野生菌株作用組均比spoT基因缺失突變菌株作用組產(chǎn)生活性氧多。 結(jié)論 H pylori spoT基因缺失突變株與野生株比較在宿主體內(nèi)定植力下降。但spoT突變株中與抗氧應(yīng)激力和生存力相關(guān)的大部分基因轉(zhuǎn)錄水平卻是上調(diào)的,毒力因子表達(dá)水平的改變及降解損傷蛋白基因的下降揭示了突變株在胃內(nèi)定植力大大降低的原因：主要是來(lái)自免疫反應(yīng)產(chǎn)生的免疫細(xì)胞對(duì)細(xì)菌的吞噬而不是spoT突變菌株抗氧應(yīng)激能力的改變。
[Abstract]:objective
Helicobacter pylori (H pylori) is an important human pathogen, gram-negative, screw like, micro aerobic bacteria, specifically parasitic on the gastric mucosa, and its infection can lead to chronic gastritis, peptic ulcers, gastric mucosa associated lymphoid tissue (MALT) lymphoma and gastric cancer. In 1994, WHO International Cancer Research The IARC has been listed as a class I carcinogen. About half of the world's population is now infected by H pylori, China is a high incidence area of infection, and the infected people usually take the bacteria for life without antibiotic treatment. In spite of this, so far the mechanism of the transmission path and persistent infection of Hpylori is not clear and thus restricts. The prevention and treatment of Hpylori infection.
H pylori induces a strong inflammatory response during the colonization of the host gastric mucosa cells. This process stimulates the host immune cells to produce a large number of reactive oxygen molecules (ROS) and active nitrogen intermediates (RNI). Although stimulated by a large number of ROS / RNI oxidation, H pylori can successfully colonize and survive in the gastric mucosa. This shows that H pylori is a new pylori. There is an effective adaptive oxygen stress system. The study found that H pylori has more anti oxidative stress gene, but the regulation gene is less, so the regulation mechanism of H pylori anti oxidative stress is not clear. It is effective to respond to cellular immunity, especially Th1 cell immune response, in which vacA and urease genes play an important role in their immune escape. However, there is little research on the immune escape regulation mechanism of Helicobacter pylori.
The spoT gene is a global post transcriptional regulation gene, and its function was first found to be a rigorous response to the regulation of Escherichia coli. The so-called rigorous response is that the dramatic changes in gene expression patterns that occur in order to respond to amino acid hunger are called "nutritional stress responses". The change in gene expression pattern, known as "strict response", is also contained in the H pylori genome. Recent studies have found that in harsh environments, the spoT gene can also regulate the rigorous response of H pylori, but the mechanism of oxygen stress and phagocytosis in the process of colonization of the host can be regarded as a rigorous environment. Therefore, our study mainly discusses the role of the spoT gene in H pylori in the host immune response to host long-term colonization, and gives a preliminary explanation of the observed phenomena, providing a clue to the prevention and treatment of Hpylori.
Method
1, the H pylori spoT gene deletion mutant and the wild strain which had been constructed were infected with Mongolia gerbil several times, respectively. Mongolia gerbil was killed at the last 2,4,6,8 week after the last infection. The gastric mucosa tissues were isolated and cultured and the bacteria were identified by Gram staining. The real time fluorescence quantitative PCR absolute quantitative method was applied to H pylori. Conduct identification and quantitative determination.
2, the immune cells in the peripheral blood were used to stimulate the H pylori spoT gene deletion mutant and the wild strain, and take two action time points: 30 minutes and 60 minutes. The sample RNA was extracted and the anti oxidative stress related genes (sodB, katA, tasA, rocF) were measured and compared after reverse transcription, and the virulence genes (cagA, vacA, napA) were reduced. The expression of clpP (clpX) at mRNA level.
3, when Mongolia gerbil was killed in the animal experiment, the blood was picked at the same time at the same time. The serum of each group was collected at -80 C. The enzyme linked immunosorbent assay was used to determine and compare the content of 8- hydroxyl deoxy guanosine (8-OHdG), a marker of DNA oxidative damage in the peripheral blood, in order to respond to the level of living oxygen in the body. The level of immune cells is induced by the infection of the same strain.
4, the immune cells of human peripheral blood were loaded with the superoxide anion fluorescence detection probe DHE. The Hpylori spoT gene deletion mutant and the wild strain acted on the immune cells of a certain amount of human peripheral blood for different time, and the reactive oxygen species were measured in vitro by flow cytometry.
Result
1, animal experiments showed that H pylori spoT gene deletion mutants and wild plants could be successfully planted in the stomach of Mongolia gerbil; the colonization ability of H pylori spoT gene deletion mutant in the gastric mucosa of Mongolia gerbil was significantly lower than that of the wild strain, and the mutant plants were not even detected at the time of eighth weeks.
2, most of the spoT gene deletion mutant strains (sodB, katA, tasA, rocF), the virulence gene (cagA, vacA, napA) were expressed at the mRNA level, and the two key genes (clpP, clpX) related to the degradation of damaged proteins were reduced.
3, the Mongolia gerbil infected H pylori wild strain group (group W) and the infected mutant group (M group), the 8-OHdG content in the peripheral blood of Mongolia gerbil in the last week after the last infection was not significant, but the eighth week M group was significantly less than that of the W group.
4, at different time points, it was found that the H pylori wild strain group produced more reactive oxygen species than the spoT gene deletion mutant strain group.
conclusion
The H pylori spoT gene deletion mutant and the wild plant were compared with the wild plant, but most of the gene transcriptional levels in the spoT mutant were up-regulated. The change of the expression level of the virulence factor and the decline of the degrading protein gene revealed that the mutagenesis was greatly reduced in the stomach. The reason is low: it is mainly from the immune reaction of immune cells that engulf bacteria rather than the ability of spoT mutant to resist oxidative stress.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378.9

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本文編號(hào)：1933751