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

【摘要】： 目的 幽門螺桿菌(Helicobacter pylori, H pylori)是一個重要的人類病原菌,革蘭染色陰性、螺桿狀、微需氧菌,特異性寄生于胃粘膜,其感染可導致慢性胃炎、消化性潰瘍、胃粘膜相關淋巴組織(MALT)淋巴瘤及胃癌的發(fā)生,1994年世界衛(wèi)生組織國際癌癥研究機構(gòu)(IARC)已將其列為Ⅰ類致癌因子。現(xiàn)在世界人口中大約一半被H pylori感染,中國是感染的高發(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)性感染機制還不明確,由此制約了針對Hpylori感染的預防和治療。 H pylori在定植于宿主胃粘膜細胞的過程中可誘發(fā)強烈的炎癥反應,這一過程刺激宿主免疫細胞產(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。這說明H pylori具有有效的適應氧應激系統(tǒng)。研究發(fā)現(xiàn)H pylori具有較多抗氧應激的效應基因,但調(diào)控基因卻較少,因此H pylori抗氧應激調(diào)控機制尚不明確。研究表明宿主通過細胞免疫防御幽門螺桿菌感染,特別是巨噬細胞的吞噬作用,然而幽門螺桿菌可以有效的應對細胞免疫特別是Th1細胞免疫應答,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)控機制卻研究甚少。 spoT基因是一個全局性的轉(zhuǎn)錄后調(diào)控基因,其功能最早發(fā)現(xiàn)是調(diào)控大腸桿菌的嚴謹應答。所謂嚴謹應答是指細菌為了應對氨基酸饑餓而發(fā)生的基因表達模式的劇烈變化也稱作“營養(yǎng)應激應答”。現(xiàn)在將細菌在各種不利生存環(huán)境下發(fā)生的基因表達模式的改變統(tǒng)稱為“嚴謹應答”。在H pylori基因組中也含有這個基因,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的嚴謹應答,但機制未明。Hpylori在宿主體內(nèi)定植過程中受到的氧應激與吞噬可以看做一種嚴謹環(huán)境,因此我們的研究主要探討spoT基因在H pylori在宿主體長期定植適應宿主免疫應答過程中所起的作用,并對觀察到的現(xiàn)象作出初步解釋,為Hpylori的預防和治療提供線索。 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,取其胃粘膜組織進行分離培養(yǎng)和革蘭染色初步鑒定細菌,應用實時熒光定量PCR絕對定量方法對H pylori進行鑒定和數(shù)量測定。 2、用人外周血中免疫細胞分別刺激H pylori spoT基因缺失突變株和野生株,并取兩個作用時間點：30分鐘和60分鐘。提取標本RNA,反轉(zhuǎn)錄后應用實時熒光定量PCR分別測定比較它們抗氧應激相關基因(sodB、katA、tasA、rocF)、毒力基因(cagA、vacA、napA)、降解受損蛋白相關基因(clpP、clpX)在mRNA水平的表達情況。 3、動物實驗中處死蒙古沙鼠時同時摘眼球取血,收集每次每組血清保存于-80℃,酶聯(lián)免疫吸附測定方法測定并比較外周血液中的DNA氧化損傷標志物8-羥基脫氧鳥苷(8-OHdG)的含量,以此反應不同菌株在體內(nèi)產(chǎn)生活性氧的水平,間接反應不同菌株感染誘導產(chǎn)生免疫細胞的水平。 4、用超氧化物陰離子熒光檢測探針DHE裝載人外周血的免疫細胞,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胞不同時間后,用流式細胞術(shù)測定比較不同菌株在體外產(chǎn)生活性氧水平。 結(jié)果 1、動物實驗表明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周時甚至幾乎檢測不到突變株定植。 2、spoT基因缺失突變株中大部分抗氧應激基因(sodB、katA、tasA、rocF)、毒力基因(cagA、vacA、napA)在mRNA水平表達異常,兩個與降解受損蛋白相關的關鍵基因(clpP、clpX)表達降低。 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含量兩組相差不大,但第8周M組明顯少于W組。 4、在各個時間點進行比較發(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突變株中與抗氧應激力和生存力相關的大部分基因轉(zhuǎn)錄水平卻是上調(diào)的,毒力因子表達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：主要是來自免疫反應產(chǎn)生的免疫細胞對細菌的吞噬而不是spoT突變菌株抗氧應激能力的改變。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R378.9

【參考文獻】

相關期刊論文 前3條

1 張群;胥文春;尹一兵;朱興華;李有強;張雪梅;;clpP基因缺陷對肺炎鏈球菌毒力表達降低的研究[J];第三軍醫(yī)大學學報;2009年04期

2 遲晶,傅寶玉,九島亮治,中島滋美,服部隆則;沙土鼠幽門螺桿菌感染胃炎、胃潰瘍動物模型的建立及除菌治療前后炎癥和細胞增殖的變化[J];世界華人消化雜志;1999年07期

3 嚴杰,胡愛萍,李強,方海偉,張苗尊;蒙古沙鼠幽門螺桿菌感染動物模型的建立[J];浙江大學學報(醫(yī)學版);2003年01期

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