幽門螺桿菌外膜蛋白18識別干擾素gamma輔助幽門螺桿菌在胃部長期定植
發(fā)布時間：2018-08-28 16:39
【摘要】：目的 幽門螺桿菌(Helicobacter pylori, H. pylori)是一種單極、多鞭毛、末端鈍圓、螺旋形彎曲的革蘭氏陰性菌,定植于全世界約一半的人體內(nèi)。在大部分帶菌者體內(nèi),H. pylori感染引起的慢性胃炎是無癥狀的,但卻被認(rèn)為是十二指腸潰瘍、胃潰瘍、胃癌及黏膜相關(guān)淋巴組織(MALT)淋巴瘤的主要致病因素。在不使用抗生素的情況下,H. pylori能夠持續(xù)定植于人體胃組織,表明宿主免疫反應(yīng)無效。同時,H. pylori耐藥率不斷升高,多數(shù)抗生素已經(jīng)無法控制H. pylori感染的蔓延,因此,對于H. pylori免疫逃逸機制的研究顯得尤為重要。 H. pylori感染誘導(dǎo)胃竇部黏膜的慢性及活動性炎癥反應(yīng),并伴隨有B細(xì)胞、T細(xì)胞及中性粒細(xì)胞的浸潤。雖然所有的H. pylori菌株都能夠引起胃炎,但是與cagA-的菌株相比,cagA+的H. pylori感染大大提高了嚴(yán)重性胃炎,萎縮性胃炎及胃癌的發(fā)病率,而且cagA+的H. pylori引起更強烈的IL-8的分泌。胃上皮細(xì)胞在急慢性H. pylori感染的過程中起重要作用,H. pylori能夠激活胃上皮細(xì)胞的多種信號轉(zhuǎn)導(dǎo)通路,激活信號通路的結(jié)果之一就是產(chǎn)生大量的IL-8.H. pylori引起的慢性活動性胃炎的胃黏膜組織中大量中性粒細(xì)胞的浸潤被認(rèn)為是對H.pylori的中性粒誘導(dǎo)因子(尿素酶,Nap A)的應(yīng)答。 IFN-γ能夠誘導(dǎo)炎癥反應(yīng),保護(hù)機體抵抗細(xì)菌感染。IFN-γ激活免疫系統(tǒng)的多個環(huán)節(jié)包括吞噬作用及抗原提呈,誘導(dǎo)抗原提呈細(xì)胞MHCⅡ的表達(dá)并激活巨噬細(xì)胞及自然殺傷細(xì)胞,因此IFN-γ在免疫反應(yīng)中起重要作用。信號傳導(dǎo)及轉(zhuǎn)錄激活因子1(STAT1)是IFN-γ信號通路的重要分子,IFN-γ能夠激活STAT1并提高其磷酸化水平,p-STAT1激活一氧化氮合酶(iNOS)并促進(jìn)一氧化氮(NO)的產(chǎn)生。NO作為IFN-γ信號通路的效應(yīng)分子,是宿主抵抗病原茵感染的基礎(chǔ)分子,能夠抑制對數(shù)期病原菌的生長。 病原菌具有能夠與宿主相接觸的多種表面結(jié)構(gòu)(包括菌毛,鞭毛,外膜蛋白以及多種分泌系統(tǒng))用以調(diào)節(jié)細(xì)菌對宿主細(xì)胞的黏附及入侵。革蘭陰性菌外膜蛋白是一種復(fù)雜的結(jié)構(gòu),主要功能是輔助細(xì)菌適應(yīng)多變的外部環(huán)境。外膜蛋白能夠主動的,有選擇性的控制重要物質(zhì)(多肽或蛋白質(zhì),核酸以及脂質(zhì)、多糖等)的流入及排出。編碼H. pylori外膜蛋白的基因約有33種,大多數(shù)外膜蛋白位于膜的表面,其主要功能包括對宿主細(xì)胞的粘附及攝取營養(yǎng)物質(zhì)。 雖然H. pylori引起強烈的炎癥反應(yīng)但是宿主免疫系統(tǒng)不能夠清除該菌,表明H. pylori具有免疫逃逸機制。H. pylori免疫逃逸機制包括誘導(dǎo)極化的免疫應(yīng)答、調(diào)節(jié)吞噬作用及中性粒細(xì)胞的功能以及抑制淋巴細(xì)胞增值。研究表明,H. pylori能夠結(jié)合IFN-γ并下調(diào)主要毒力因子CagA的表達(dá)。但是目前尚未有H. pylori結(jié)合IFN-γ的具體機制的相關(guān)報道,本論文通過基因芯片篩選出H. pylori外膜蛋白Omp18,并進(jìn)一步證實Omp18能夠結(jié)合IFN-γ輔助H. pylori長期定植,有助于提高H. pylori在NO壓力下的生存力及抗吞噬作用。 方法 1.通過基因芯片方法篩選出H. pylori野生株在IFN-γ作用下發(fā)生表達(dá)變化的基因。用IFN-γ處理培養(yǎng)至對數(shù)期的H. pylori野生株8h,未處理組作為對照,分別進(jìn)行基因芯片檢測,通過比較找出IFN-γ作用下發(fā)生表達(dá)變化的基因。 2.通過Real-Time PCR法驗證基因芯片結(jié)果。用IFN-γ處理培養(yǎng)至對數(shù)期的H.pylori野生株,分別于0h,2h,4h,8h收集菌液,提取RNA反轉(zhuǎn)錄為cDNA,通過Real-Time PCR檢測omp18的表達(dá)。 3.檢測H. pylori野生株毒力因子CagA\NapA在IFN-γ作用下的表達(dá)情況。H.pylori野生株及omp18突變株過夜培養(yǎng)至對數(shù)生長期,用IFN-γ處理8h,未處理組作為對照,收集菌液分別用于提取細(xì)菌總蛋白及RNA,通過western blot及Real-Time PCR檢測毒力因子CagA、NapA的表達(dá)情況。 4.動物實驗。將H. pylori野生株及ompl8突變株分別多次經(jīng)口感染蒙古沙鼠,分別于末次感染后第2,4,6,8周脫頸處死蒙古沙鼠,取其胃竇組織進(jìn)行細(xì)菌的分離培養(yǎng)檢測H. pylori定植情況,并通過HE染色鑒定胃組織炎癥狀況,應(yīng)用Real-Time PCR及ELISA檢測胃組織炎性因子表達(dá)情況。 5.檢測H. pylori野生株及ompl8突變株生存能力的差異。 (1)用硝普鈉(SNP)處理培養(yǎng)至對數(shù)期的H. pylori野生株及omp18突變株,分別通過瓊脂稀釋法及熒光染色法比較H. pylori野生株及omp18突變株的生存力。 (2)將培養(yǎng)至對數(shù)生長期的H. pylori野生株及omp18突變株分別加入至培養(yǎng)巨噬細(xì)胞的孔板中,分別于第2h,6h,24h裂解巨噬細(xì)胞并將含有H. pylori的裂解液經(jīng)梯度稀釋后涂布至瓊脂平板上,培養(yǎng)3-5天后計數(shù)單克隆。 6.通過免疫熒光的方法證實H. pylori外膜蛋白Omp18能夠結(jié)合IFN-γ。 7.T-COFFEE軟件分析Omp18,OprF及干擾素結(jié)合區(qū)域蛋白序列。 8.通過western blot比較H. pylori野生株與omp18突變株刺激后巨噬細(xì)胞p-STAT1蛋白的表達(dá)差異。 9.NO測定。將H. pylori感染后的巨噬細(xì)胞及沙鼠胃組織分泌的亞硝酸鹽的含量作為NO分泌的間接指標(biāo)并通過Griess反應(yīng)測定。 結(jié)果 1.IFN-y刺激下H. pylori Omp18表達(dá)升高�；蛐酒l(fā)現(xiàn)與未處理的菌株相比,經(jīng)10ng/mL IFN-γ處理后H. pylori野生株Omp18表達(dá)升高。 2.T-COFFEE軟件分析發(fā)現(xiàn)Ompl8序列與OprF序列以及干擾素gamma感受器1(IRF1)的干擾素結(jié)合域相似度很高。 3.免疫熒光證實IFN-γ能夠與H. pylori Omp18結(jié)合。 4.IFN-γ下調(diào)H. pylori野生株毒力因子的表達(dá)。Western blot及Real-Time PCR發(fā)現(xiàn)經(jīng)10ng/mL IFN-γ處理后H. pylori野生株CagA及NapA表達(dá)下降,而ompl8突變株中CagA及NapA表達(dá)上升。 5.H. pylori omp18突變株在蒙古沙鼠胃組織中存在定植缺陷。動物實驗表明H.pylori野生株及ompl8突變株均可在蒙古沙鼠胃部定植；ompl8突變株在蒙古沙鼠胃組織的定植力明顯低于野生株,第8周差異最明顯。 6.H. pylori omp18突變株感染誘導(dǎo)強烈的炎癥反應(yīng)。與野生株感染的蒙古沙鼠相比,omp18突變株感染的蒙古沙鼠胃組織表現(xiàn)出更多的中性粒細(xì)胞浸潤及更嚴(yán)重的組織損傷；Real-Time PCR及ELISA檢測發(fā)現(xiàn)omp18突變株誘導(dǎo)蒙古沙鼠胃組織分泌更多的炎性因子；同樣,omp18突變株感染誘導(dǎo)AGS細(xì)胞分泌更多IL-8,誘導(dǎo)巨噬細(xì)胞分泌更多MIP-2,特別是在IFN-γ存在時。 7.H. pylori omp18突變株感染上調(diào)NO表達(dá)。在10ng/mL IFN-γ作用下,H. pylori野生株而非omp18突變株能夠抑制巨噬細(xì)胞p-STAT1的表達(dá)；而omp18突變株能夠誘導(dǎo)巨噬細(xì)胞以及蒙古沙鼠胃組織NO高表達(dá)。 8.Omp18有助于提高H. pylori野生株在氧應(yīng)激下的生存力及抗吞噬作用。在SNP刺激下,與H. pylori野生株相比,omp18突變株生存力急劇下降,且絕大多數(shù)突變株由正常的螺桿狀變?yōu)榍蛐�；同�?omp18突變株在巨噬細(xì)胞中生存力減弱。 結(jié)論 H. pylori能夠通過Omp18主動感應(yīng)IFN-y的變化,優(yōu)化自身基因型從而避免誘導(dǎo)強烈的免疫應(yīng)答以期達(dá)到長期定植。另外,Omp18有助于提高H. pylori在NO壓力下的生存力及抗吞噬作用,為H. pylori在宿主胃部的長期定植奠定了基礎(chǔ)。
[Abstract]:objective
Helicobacter pylori (H. pylori) is a unipolar, multi-flagellated, obtuse-rounded, spirally curved Gram-negative bacterium that colonizes about half of the world's population. The main pathogenic factor of mucosa-associated lymphoid tissue (MALT) lymphoma. H.pylori can persist in human gastric tissues without antibiotics, indicating that the host immune response is ineffective. At the same time, H.pylori resistance rate is increasing, most antibiotics have been unable to control the spread of H.pylori infection, therefore, for H.pylori immune escape. The study of escape mechanism is particularly important.
H. pylori infection induces chronic and active inflammation of the antral mucosa with infiltration of B, T and neutrophils. Although all H. pylori strains can cause gastritis, H. pylori infection with cagA + significantly increases the incidence of severe gastritis, atrophic gastritis and gastric cancer compared with cagA - strains. Gastric epithelial cells play an important role in the process of acute and chronic H. pylori infection. H. pylori can activate a variety of signal transduction pathways in gastric epithelial cells. One of the results of activating signal pathways is the production of a large number of IL-8.H. pylori-induced chronic active gastritis gastric mucosa. Invasion of large numbers of neutrophils in tissues is thought to be a response to H.pylori's neutrophil-inducible factor (urease, Nap A).
IFN-gamma can induce inflammation and protect the body against bacterial infection. IFN-gamma activates many aspects of the immune system, including phagocytosis and antigen presentation, induces the expression of antigen-presenting cells MHC II and activates macrophages and natural killer cells. Therefore, IFN-gamma plays an important role in the immune response. Signal transduction and activator of transcription 1 (S-1) TAT1 is an important molecule in the IFN-gamma signaling pathway. IFN-gamma can activate STAT1 and increase its phosphorylation level. p-STAT1 activates nitric oxide synthase (iNOS) and promotes the production of nitric oxide (NO). As an effector molecule of the IFN-gamma signaling pathway, NO is the basic molecule of host resistance to pathogenic infection and can inhibit the growth of logarithmic pathogenic bacteria.
Pathogens have a variety of surface structures (including fimbriae, flagella, adventitia proteins and secretory systems) that can interact with host cells to regulate bacterial adhesion and invasion. Gram-negative bacteria adventitia proteins are complex structures whose main function is to assist bacteria to adapt to changeable external environments. There are about 33 genes encoding H.pylori outer membrane proteins. Most outer membrane proteins are located on the surface of the membrane. Their main functions include adhesion to host cells and uptake of nutrients.
Although H.pylori induces a strong inflammatory response, the host immune system is unable to clear the bacteria, suggesting that H.pylori has an immune escape mechanism. H.pylori immune escape mechanism includes induced polarized immune response, regulation of phagocytosis, neutrophil function and inhibition of lymphocyte proliferation. IFN-gamma also down-regulates the expression of CagA, a major virulence factor. However, no specific mechanism of H.pylori binding to IFN-gamma has been reported. In this study, the outer membrane protein Omp18 of H.pylori was screened by gene chip, and further confirmed that Omp18 can bind to IFN-gamma to assist H.pylori long-term colonization, which is helpful to improve H.pylori's survival under NO pressure. Force and anti phagocytosis.
Method
1. The genes of H. pylori wild strains were screened out by gene chip. The H. pylori wild strains were incubated with IFN-gamma for 8 hours. The genes of H. pylori wild strains were detected by gene chip and compared with the untreated ones.
2. The results of gene chip were verified by Real-Time PCR. The H.pylori wild strains were cultured in logarithmic phase by IFN-gamma treatment. The bacterial liquids were collected at 0 h, 2 h, 4 h and 8 h respectively. RNA was retrieved and transcribed into cDNA. The expression of omp18 was detected by Real-Time PCR.
3. To detect the expression of CagANapA in H. pylori wild strain under the action of IFN-gamma. H. pylori wild strain and omp18 mutant were overnight cultured to logarithmic growth stage, treated with IFN-gamma for 8 hours, and untreated as control. The bacterial liquor was collected for extracting total bacterial protein and RNA, and the virulence factors CagA, Na and Real-Time PCR were detected by Western blot and Real-Time PCR. The expression of pA.
4. Animal experiment. Mongolian gerbils were orally infected with H. pylori wild strain and ompl8 mutant for many times, and were killed at the 2nd, 4th, 6th and 8th weeks after the last infection. The gastric antrum tissues of Mongolian gerbils were isolated and cultured to detect the colonization of H. pylori, and the gastric inflammation was identified by HE staining. Real-Time PCR and ELISA were used to detect the gastric inflammation. The expression of inflammatory factors in gastric tissue was detected.
5. to detect the difference in viability between H. pylori wild strain and ompl8 mutant strain.
(1) H. pylori wild strain and omp18 mutant strain were treated with sodium nitroprusside (SNP) and cultured to logarithmic phase. The viability of H. pylori wild strain and omp18 mutant strain were compared by agar dilution and fluorescence staining respectively.
(2) H. pylori wild strain and omp18 mutant strain were added to the pore plate of cultured macrophages at logarithmic growth stage. Macrophages were lysed at 2h, 6h and 24h respectively, and the lysate containing H. pylori was coated on agar plate after gradient dilution. Monoclones were counted after 3-5 days of culture.
6. immunofluorescence assay confirmed that H. pylori outer membrane protein Omp18 could bind IFN- gamma.
7.T-COFFEE software was used to analyze Omp18, OprF and IFN binding region protein sequences.
8. The expression of p-STAT1 protein in macrophages stimulated by H.pylori wild strain and omp18 mutant was compared by Western blot.
The nitrite secreted by macrophages and gastric tissues of gerbils infected with H.pylori was used as an indirect indicator of NO secretion and determined by Griess reaction.
Result
1. The expression of H. pylori Omp18 increased under IFN-y stimulation. Compared with untreated strains, the expression of H. pylori Omp18 increased after 10 ng/mL IFN-gamma treatment.
2. T-COFFEE software analysis found that the Ompl8 sequence was highly similar to the OprF sequence and the interferon binding domain of interferon gamma receptor 1 (IRF1).
3. immunofluorescence confirmed that IFN- gamma could bind to H. pylori Omp18.
4. IFN-gamma down-regulated the expression of virulence factors in H. pylori wild strain. Western blot and Real-time PCR showed that the expression of CagA and NapA in H. pylori wild strain decreased after 10 ng/mL IFN-gamma treatment, while the expression of CagA and NapA in ompl8 mutant increased.
5. H. pylori omp18 mutant was found to be defective in the stomach of Mongolian gerbils. Animal experiments showed that both H. pylori wild strain and ompl8 mutant could be colonized in the stomach of Mongolian gerbils. The colonization ability of ompl8 mutant in the stomach of Mongolian gerbils was significantly lower than that of wild strain, and the difference was most obvious at the 8th week.
6. H. pylori omp18 mutant infection induced strong inflammation. Compared with Mongolian gerbils infected with wild strain, the gastric tissue of Mongolian gerbils infected with omp18 mutant showed more neutrophil infiltration and more serious tissue damage; Real-Time PCR and ELISA detection showed that the gastric tissue of Mongolian gerbils was secreted more by omp18 mutant. Similarly, infection with omp18 mutant induces AGS cells to secrete more IL-8 and macrophages to secrete more MIP-2, especially in the presence of IFN-gamma.
7.H.pylori omp18 mutant infection up-regulated the expression of NO.Under the effect of 10ng/mL IFN-gamma, H.pylori wild strain, not omp18 mutant, could inhibit the expression of p-STAT1 in macrophages, while omp18 mutant could induce the high expression of NO in macrophages and Mongolian gerbil gastric tissue.
8.Omp18 is helpful to improve the viability and anti-phagocytosis of H.pylori wild strain under oxygen stress.Compared with H.pylori wild strain under SNP stimulation, the viability of omp18 mutant decreased sharply, and most of the mutants changed from normal screw to sphere.Similarly, the viability of omp18 mutant in macrophages decreased.
conclusion
H.pylori can actively induce changes in IFN-y by Omp18 to optimize its genotype and avoid inducing strong immune responses in order to achieve long-term colonization.In addition, Omp18 helps to improve H.pylori's viability and anti-phagocytosis under NO stress, laying the foundation for H.pylori's long-term colonization in the stomach of the host.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R573
[Abstract]:objective
Helicobacter pylori (H. pylori) is a unipolar, multi-flagellated, obtuse-rounded, spirally curved Gram-negative bacterium that colonizes about half of the world's population. The main pathogenic factor of mucosa-associated lymphoid tissue (MALT) lymphoma. H.pylori can persist in human gastric tissues without antibiotics, indicating that the host immune response is ineffective. At the same time, H.pylori resistance rate is increasing, most antibiotics have been unable to control the spread of H.pylori infection, therefore, for H.pylori immune escape. The study of escape mechanism is particularly important.
H. pylori infection induces chronic and active inflammation of the antral mucosa with infiltration of B, T and neutrophils. Although all H. pylori strains can cause gastritis, H. pylori infection with cagA + significantly increases the incidence of severe gastritis, atrophic gastritis and gastric cancer compared with cagA - strains. Gastric epithelial cells play an important role in the process of acute and chronic H. pylori infection. H. pylori can activate a variety of signal transduction pathways in gastric epithelial cells. One of the results of activating signal pathways is the production of a large number of IL-8.H. pylori-induced chronic active gastritis gastric mucosa. Invasion of large numbers of neutrophils in tissues is thought to be a response to H.pylori's neutrophil-inducible factor (urease, Nap A).
IFN-gamma can induce inflammation and protect the body against bacterial infection. IFN-gamma activates many aspects of the immune system, including phagocytosis and antigen presentation, induces the expression of antigen-presenting cells MHC II and activates macrophages and natural killer cells. Therefore, IFN-gamma plays an important role in the immune response. Signal transduction and activator of transcription 1 (S-1) TAT1 is an important molecule in the IFN-gamma signaling pathway. IFN-gamma can activate STAT1 and increase its phosphorylation level. p-STAT1 activates nitric oxide synthase (iNOS) and promotes the production of nitric oxide (NO). As an effector molecule of the IFN-gamma signaling pathway, NO is the basic molecule of host resistance to pathogenic infection and can inhibit the growth of logarithmic pathogenic bacteria.
Pathogens have a variety of surface structures (including fimbriae, flagella, adventitia proteins and secretory systems) that can interact with host cells to regulate bacterial adhesion and invasion. Gram-negative bacteria adventitia proteins are complex structures whose main function is to assist bacteria to adapt to changeable external environments. There are about 33 genes encoding H.pylori outer membrane proteins. Most outer membrane proteins are located on the surface of the membrane. Their main functions include adhesion to host cells and uptake of nutrients.
Although H.pylori induces a strong inflammatory response, the host immune system is unable to clear the bacteria, suggesting that H.pylori has an immune escape mechanism. H.pylori immune escape mechanism includes induced polarized immune response, regulation of phagocytosis, neutrophil function and inhibition of lymphocyte proliferation. IFN-gamma also down-regulates the expression of CagA, a major virulence factor. However, no specific mechanism of H.pylori binding to IFN-gamma has been reported. In this study, the outer membrane protein Omp18 of H.pylori was screened by gene chip, and further confirmed that Omp18 can bind to IFN-gamma to assist H.pylori long-term colonization, which is helpful to improve H.pylori's survival under NO pressure. Force and anti phagocytosis.
Method
1. The genes of H. pylori wild strains were screened out by gene chip. The H. pylori wild strains were incubated with IFN-gamma for 8 hours. The genes of H. pylori wild strains were detected by gene chip and compared with the untreated ones.
2. The results of gene chip were verified by Real-Time PCR. The H.pylori wild strains were cultured in logarithmic phase by IFN-gamma treatment. The bacterial liquids were collected at 0 h, 2 h, 4 h and 8 h respectively. RNA was retrieved and transcribed into cDNA. The expression of omp18 was detected by Real-Time PCR.
3. To detect the expression of CagANapA in H. pylori wild strain under the action of IFN-gamma. H. pylori wild strain and omp18 mutant were overnight cultured to logarithmic growth stage, treated with IFN-gamma for 8 hours, and untreated as control. The bacterial liquor was collected for extracting total bacterial protein and RNA, and the virulence factors CagA, Na and Real-Time PCR were detected by Western blot and Real-Time PCR. The expression of pA.
4. Animal experiment. Mongolian gerbils were orally infected with H. pylori wild strain and ompl8 mutant for many times, and were killed at the 2nd, 4th, 6th and 8th weeks after the last infection. The gastric antrum tissues of Mongolian gerbils were isolated and cultured to detect the colonization of H. pylori, and the gastric inflammation was identified by HE staining. Real-Time PCR and ELISA were used to detect the gastric inflammation. The expression of inflammatory factors in gastric tissue was detected.
5. to detect the difference in viability between H. pylori wild strain and ompl8 mutant strain.
(1) H. pylori wild strain and omp18 mutant strain were treated with sodium nitroprusside (SNP) and cultured to logarithmic phase. The viability of H. pylori wild strain and omp18 mutant strain were compared by agar dilution and fluorescence staining respectively.
(2) H. pylori wild strain and omp18 mutant strain were added to the pore plate of cultured macrophages at logarithmic growth stage. Macrophages were lysed at 2h, 6h and 24h respectively, and the lysate containing H. pylori was coated on agar plate after gradient dilution. Monoclones were counted after 3-5 days of culture.
6. immunofluorescence assay confirmed that H. pylori outer membrane protein Omp18 could bind IFN- gamma.
7.T-COFFEE software was used to analyze Omp18, OprF and IFN binding region protein sequences.
8. The expression of p-STAT1 protein in macrophages stimulated by H.pylori wild strain and omp18 mutant was compared by Western blot.
The nitrite secreted by macrophages and gastric tissues of gerbils infected with H.pylori was used as an indirect indicator of NO secretion and determined by Griess reaction.
Result
1. The expression of H. pylori Omp18 increased under IFN-y stimulation. Compared with untreated strains, the expression of H. pylori Omp18 increased after 10 ng/mL IFN-gamma treatment.
2. T-COFFEE software analysis found that the Ompl8 sequence was highly similar to the OprF sequence and the interferon binding domain of interferon gamma receptor 1 (IRF1).
3. immunofluorescence confirmed that IFN- gamma could bind to H. pylori Omp18.
4. IFN-gamma down-regulated the expression of virulence factors in H. pylori wild strain. Western blot and Real-time PCR showed that the expression of CagA and NapA in H. pylori wild strain decreased after 10 ng/mL IFN-gamma treatment, while the expression of CagA and NapA in ompl8 mutant increased.
5. H. pylori omp18 mutant was found to be defective in the stomach of Mongolian gerbils. Animal experiments showed that both H. pylori wild strain and ompl8 mutant could be colonized in the stomach of Mongolian gerbils. The colonization ability of ompl8 mutant in the stomach of Mongolian gerbils was significantly lower than that of wild strain, and the difference was most obvious at the 8th week.
6. H. pylori omp18 mutant infection induced strong inflammation. Compared with Mongolian gerbils infected with wild strain, the gastric tissue of Mongolian gerbils infected with omp18 mutant showed more neutrophil infiltration and more serious tissue damage; Real-Time PCR and ELISA detection showed that the gastric tissue of Mongolian gerbils was secreted more by omp18 mutant. Similarly, infection with omp18 mutant induces AGS cells to secrete more IL-8 and macrophages to secrete more MIP-2, especially in the presence of IFN-gamma.
7.H.pylori omp18 mutant infection up-regulated the expression of NO.Under the effect of 10ng/mL IFN-gamma, H.pylori wild strain, not omp18 mutant, could inhibit the expression of p-STAT1 in macrophages, while omp18 mutant could induce the high expression of NO in macrophages and Mongolian gerbil gastric tissue.
8.Omp18 is helpful to improve the viability and anti-phagocytosis of H.pylori wild strain under oxygen stress.Compared with H.pylori wild strain under SNP stimulation, the viability of omp18 mutant decreased sharply, and most of the mutants changed from normal screw to sphere.Similarly, the viability of omp18 mutant in macrophages decreased.
conclusion
H.pylori can actively induce changes in IFN-y by Omp18 to optimize its genotype and avoid inducing strong immune responses in order to achieve long-term colonization.In addition, Omp18 helps to improve H.pylori's viability and anti-phagocytosis under NO stress, laying the foundation for H.pylori's long-term colonization in the stomach of the host.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R573
【共引文獻(xiàn)】
相關(guān)期刊論文 前10條
1 梁坤;張紅珠;周廣璽;孫向紅;王輝明;張翠萍;;逆萎康對慢性萎縮性胃炎大鼠胃黏膜組織P16和TGF-β1表達(dá)影響[J];青島大學(xué)醫(yī)學(xué)院學(xué)報;2014年01期
2 秦李娜;劉英;胡海峰;;聚乙二醇修飾的精氨酸脫亞胺酶臨床研究[J];世界臨床藥物;2014年04期
3 Jun Wang;Wenyi Qian;Qing Zhu;Jian Chen;Fei Huan;Rong Gao;Hang Xiao;;Martentoxin, a large-conductance Ca~(2+)-activated K~+ channel inhibitor, attenuated TNF-α-induced nitric oxide release by human umbilical vein endothelial cells[J];The Journal of Biomedical Research;2013年05期
4 陳娜;莊園;王婷婷;彭六生;程平;李文華;趙永亮;余佩武;鄒全明;;肥大細(xì)胞在胃癌患者中分布及其免疫抑制作用的研究[J];免疫學(xué)雜志;2014年01期
5 謝鐵松;雷如軍;邵義敏;楊靖;劉益國;;幽門螺桿菌在胃癌形成中的作用機制[J];四川生理科學(xué)雜志;2014年01期
6 謝玲;郭新異;張nQ澤;龐礴;都玉蓉;鄒小艷;郭松長;趙新全;;高原鼠兔誘導(dǎo)型一氧化氮合酶cDNA克隆及序列分析[J];四川動物;2014年04期
7 李巧珍;陳玉強;顏江華;丁園;王勇軍;王生育;葉燕華;許英藝;何蕙香;;幽門螺桿菌感染對胃癌患者外周血MODCs功能的影響[J];中國腫瘤臨床;2013年15期
8 凌峰;王曉春;陳s，
本文編號：2209970
本文鏈接：http://sikaile.net/yixuelunwen/xiaohjib/2209970.html
最近更新
教材專著
