spoT基因在幽门螺杆菌定植过程中的功能研究

发布时间：2018-05-25 15:33

本文选题：spoT基因 + 幽门螺杆菌　；参考：《山东大学》2010年硕士论文

【摘要】： 目的幽门螺杆菌(Helicobacter pylori, H pylori)是一个重要的人类病原菌,革兰染色阴性、螺杆状、微需氧菌,特异性寄生于胃粘膜,其感染可导致慢性胃炎、消化性溃疡、胃粘膜相关淋巴组织(MALT)淋巴瘤及胃癌的发生,1994年世界卫生组织国际癌症研究机构(IARC)已将其列为Ⅰ类致癌因子。现在世界人口中大约一半被H pylori感染,中国是感染的高发区,并且在不接受抗菌治疗的情况下被感染者一般终生带菌。尽管如此,迄今为止Hpylori的传播途径和持续性感染机制还不明确,由此制约了针对Hpylori感染的预防和治疗。 H pylori在定植于宿主胃粘膜细胞的过程中可诱发强烈的炎症反应,这一过程刺激宿主免疫细胞产生大量的活性氧分子(ROS)和活性氮中介物(RNI)。虽然受到大量ROS／RNI的氧化刺激,H pylori却能够成功地在胃粘膜定植并生存。这说明H pylori具有有效的适应氧应激系统。研究发现H pylori具有较多抗氧应激的效应基因,但调控基因却较少,因此H pylori抗氧应激调控机制尚不明确。研究表明宿主通过细胞免疫防御幽门螺杆菌感染,特别是巨噬细胞的吞噬作用,然而幽门螺杆菌可以有效的应对细胞免疫特别是Th1细胞免疫应答,其中vacA与尿素酶基因在其免疫逃逸中具有重要作用。然而,幽门螺杆菌免疫逃逸调控机制却研究甚少。 spoT基因是一个全局性的转录后调控基因,其功能最早发现是调控大肠杆菌的严谨应答。所谓严谨应答是指细菌为了应对氨基酸饥饿而发生的基因表达模式的剧烈变化也称作“营养应激应答”。现在将细菌在各种不利生存环境下发生的基因表达模式的改变统称为“严谨应答”。在H pylori基因组中也含有这个基因,最近研究发现在恶劣环境下,spoT基因也可以调控H pylori的严谨应答,但机制未明。Hpylori在宿主体内定植过程中受到的氧应激与吞噬可以看做一种严谨环境,因此我们的研究主要探讨spoT基因在H pylori在宿主体长期定植适应宿主免疫应答过程中所起的作用,并对观察到的现象作出初步解释,为Hpylori的预防和治疗提供线索。方法 1、将已构建好的H pylori spoT基因缺失突变株和野生株分别多次经口感染蒙古沙鼠,分别于末次感染后的第2、4、6、8周脱颈处死蒙古沙鼠,取其胃粘膜组织进行分离培养和革兰染色初步鉴定细菌,应用实时荧光定量PCR绝对定量方法对H pylori进行鉴定和数量测定。 2、用人外周血中免疫细胞分别刺激H pylori spoT基因缺失突变株和野生株,并取两个作用时间点：30分钟和60分钟。提取标本RNA,反转录后应用实时荧光定量PCR分别测定比较它们抗氧应激相关基因(sodB、katA、tasA、rocF)、毒力基因(cagA、vacA、napA)、降解受损蛋白相关基因(clpP、clpX)在mRNA水平的表达情况。 3、动物实验中处死蒙古沙鼠时同时摘眼球取血,收集每次每组血清保存于-80℃,酶联免疫吸附测定方法测定并比较外周血液中的DNA氧化损伤标志物8-羟基脱氧鸟苷(8-OHdG)的含量,以此反应不同菌株在体内产生活性氧的水平,间接反应不同菌株感染诱导产生免疫细胞的水平。 4、用超氧化物阴离子荧光检测探针DHE装载人外周血的免疫细胞,Hpylori spoT基因缺失突变株和野生株分别作用于一定量的人外周血的免疫细胞不同时间后,用流式细胞术测定比较不同菌株在体外产生活性氧水平。结果 1、动物实验表明H pylori spoT基因缺失突变株和野生株均可在蒙古沙鼠胃内成功定植；H pylori spoT基因缺失突变株在蒙古沙鼠胃粘膜的定植能力明显低于野生株,在第8周时甚至几乎检测不到突变株定植。 2、spoT基因缺失突变株中大部分抗氧应激基因(sodB、katA、tasA、rocF)、毒力基因(cagA、vacA、napA)在mRNA水平表达异常,两个与降解受损蛋白相关的关键基因(clpP、clpX)表达降低。 3、蒙古沙鼠感染H pylori野生菌株组(W组)与感染突变菌株组(M组)比较,末次感染后第2、4、6周蒙古沙鼠外周血中8-OHdG含量两组相差不大,但第8周M组明显少于W组。 4、在各个时间点进行比较发现：H pylori野生菌株作用组均比spoT基因缺失突变菌株作用组产生活性氧多。结论 H pylori spoT基因缺失突变株与野生株比较在宿主体内定植力下降。但spoT突变株中与抗氧应激力和生存力相关的大部分基因转录水平却是上调的,毒力因子表达水平的改变及降解损伤蛋白基因的下降揭示了突变株在胃内定植力大大降低的原因：主要是来自免疫反应产生的免疫细胞对细菌的吞噬而不是spoT突变菌株抗氧应激能力的改变。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378.9

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