【摘要】： 鼠疫是由鼠疫耶爾森氏菌(以下簡(jiǎn)稱鼠疫菌)引起的一種烈性傳染病,其流行給人類帶來(lái)了巨大的災(zāi)難,因此對(duì)鼠疫菌致病機(jī)制的研究是做好鼠疫防控的重要切入點(diǎn)。鼠疫菌感染宿主引發(fā)致病是個(gè)復(fù)雜的多步驟過(guò)程。鼠疫菌在體內(nèi)致病的關(guān)鍵環(huán)節(jié)是在巨噬細(xì)胞內(nèi)的生存繁殖,該菌與巨噬細(xì)胞相互作用不僅不會(huì)被殺死,反而能夠生存,并合成大量毒力因子,最終巨噬細(xì)胞破裂,釋放出的鼠疫菌具備強(qiáng)大的抗吞噬能力,并為分泌毒力因子做好準(zhǔn)備。因此,在感染早期,鼠疫菌在宿主巨噬細(xì)胞的生存與繁殖是鼠疫菌致病的一個(gè)重要環(huán)節(jié)。鼠疫菌的毒力之一是pCD1質(zhì)粒編碼的Ⅲ型分泌系統(tǒng)(type III secretion system, T3SS),通過(guò)六個(gè)效應(yīng)蛋白輸入真核細(xì)胞從而打斷宿主的防御機(jī)制。本研究以鼠疫菌T3SS注射小體結(jié)構(gòu)蛋白YscW為研究對(duì)象,分析yscW基因缺失后對(duì)鼠疫菌毒力的影響;探究yscW突變株與互補(bǔ)株巨噬細(xì)胞效應(yīng);研究YscW蛋白是否會(huì)直接影響巨噬細(xì)胞生存及免疫調(diào)節(jié)功能;探討YscW蛋白功能的可能機(jī)制。 首先利用自殺質(zhì)粒方法構(gòu)建yscW突變株,在此基礎(chǔ)上導(dǎo)入與鼠疫菌拷貝數(shù)接近的pACYC184-YscW的重組質(zhì)粒構(gòu)建互補(bǔ)株。LD50實(shí)驗(yàn)觀察yscW對(duì)鼠疫菌致病性的影響,結(jié)果表明鼠疫菌缺失yscW基因,其致病性明顯減弱;同時(shí)體外感染巨噬細(xì)胞,顯微鏡觀察表明YscW影響巨噬細(xì)胞形態(tài)。 進(jìn)而構(gòu)建小鼠腹腔感染模型,分析鼠疫菌野生株、yscW突變株與互補(bǔ)株對(duì)巨噬細(xì)胞生存的影響,體、內(nèi)外實(shí)驗(yàn)證實(shí)yscW突變株明顯降低巨噬細(xì)胞凋亡,而互補(bǔ)株恢復(fù)了突變株導(dǎo)致的凋亡改變。通過(guò)對(duì)凋亡的進(jìn)一步分析,我們發(fā)現(xiàn)不同菌株間導(dǎo)致細(xì)胞凋亡的差異主要來(lái)源于細(xì)胞早期凋亡的改變,而對(duì)于晚期凋亡和細(xì)胞壞死無(wú)差異。此外,細(xì)胞周期的分析顯示,三株菌無(wú)顯著差異。同時(shí),凋亡誘導(dǎo)關(guān)鍵蛋白Caspase-3的表達(dá)檢測(cè)結(jié)果與三株菌感染后細(xì)胞水平凋亡變化相一致,從分子水平證實(shí)凋亡差異。然而過(guò)表達(dá)YscW至RAW264.7細(xì)胞后,實(shí)驗(yàn)組與對(duì)照組細(xì)胞凋亡與細(xì)胞周期間無(wú)顯著差異,表明YscW并不是誘導(dǎo)細(xì)胞凋亡的直接效應(yīng)蛋白。YopJ是鼠疫菌Ⅲ型分泌系統(tǒng)六個(gè)效應(yīng)蛋白中唯一與細(xì)胞凋亡相關(guān)的蛋白,為了研究凋亡差異的機(jī)制,對(duì)三株菌YopJ分泌進(jìn)行檢測(cè),結(jié)果表明yscW突變導(dǎo)致YopJ分泌減少。因此,鼠疫菌yscW突變株YopJ分泌減少是導(dǎo)致該突變株誘導(dǎo)細(xì)胞凋亡減少的機(jī)制之一。 對(duì)于不同鼠疫菌株感染后巨噬細(xì)胞的功能改變進(jìn)行深入研究,結(jié)果顯示鼠疫菌YscW影響了巨噬細(xì)胞的免疫調(diào)節(jié)功能。吞噬功能、抗原提呈能力和細(xì)胞因子分泌是巨噬細(xì)胞的三大主要功能。以雞紅細(xì)胞作為靶細(xì)胞,分析不同菌株感染后巨噬細(xì)胞吞噬功能的變化,結(jié)果顯示無(wú)論野生株、突變株還是互補(bǔ)株,感染后巨噬細(xì)胞的吞噬功能明顯低于正常巨噬細(xì)胞,表明鼠疫菌感染后,宿主巨噬細(xì)胞吞噬功能降低;而感染鼠疫菌yscW突變株的細(xì)胞,與野生株和互補(bǔ)株感染比較,其吞噬功能又明顯增強(qiáng),說(shuō)明yscW基因直接或間接的參與了病原菌調(diào)節(jié)宿主細(xì)胞的吞噬反應(yīng)。進(jìn)而過(guò)表達(dá)YscW的細(xì)胞,其吞噬功能明顯低于對(duì)照組細(xì)胞,更直接的給出了YscW直接調(diào)節(jié)巨噬細(xì)胞吞噬功能的證據(jù)。巨噬細(xì)胞對(duì)OVA(卵白蛋白)抗原小肽的提呈能力通過(guò)混合淋巴細(xì)胞培養(yǎng),T細(xì)胞增殖程度加以判定。結(jié)果顯示突變株感染后的巨噬細(xì)胞抗原提呈能力明顯高于同樣條件下野生株感染的細(xì)胞,而過(guò)表達(dá)YscW的細(xì)胞其抗原提呈能力又顯著低于對(duì)照組細(xì)胞,這些結(jié)果表明YscW直接影響了宿主巨噬細(xì)胞的抗原提呈能力。此外,感染后巨噬細(xì)胞及過(guò)表達(dá)細(xì)胞表面分子的檢測(cè),進(jìn)一步證實(shí)YscW對(duì)細(xì)胞抗原提呈能力的影響。巨噬細(xì)胞體外感染鼠疫菌后,細(xì)胞分泌的TNF-α水平明顯高于未受到細(xì)菌刺激的正常細(xì)胞;yscW突變株感染后巨噬細(xì)胞TNF-α分泌顯著高于其它兩株菌,表明yscW基因缺失巨噬細(xì)胞促炎癥因子釋放增多,從影響細(xì)胞因子分泌的角度研究了YscW的作用。上述研究結(jié)果首次表明,鼠疫菌Ⅲ型分泌系統(tǒng)的結(jié)構(gòu)蛋白YscW直接參與到宿主巨噬細(xì)胞免疫調(diào)節(jié)反應(yīng)中,干擾了細(xì)胞正常的生理機(jī)能。 本實(shí)驗(yàn)室前期工作發(fā)現(xiàn)鼠疫菌YscW蛋白可能與巨噬細(xì)胞Rnf149蛋白存在相互作用,這就提示上述觀察到的YscW巨噬細(xì)胞生物學(xué)效應(yīng)是否與YscW和Rnf149的相互作用相關(guān)聯(lián)。因此對(duì)兩種蛋白相互作用的驗(yàn)證成為后續(xù)實(shí)驗(yàn)的基礎(chǔ)。對(duì)于上述兩種蛋白,利用分子克隆技術(shù)分別與多個(gè)表達(dá)載體相連,并通過(guò)鎳柱和GS-4B柱進(jìn)行蛋白的純化,最終獲得帶有GST和His標(biāo)簽的兩種蛋白。進(jìn)而GST-Pull Down實(shí)驗(yàn)體外驗(yàn)證了YscW與巨噬細(xì)胞靶蛋白R(shí)nf149結(jié)合,為進(jìn)一步的體內(nèi)驗(yàn)證奠定基礎(chǔ)。 本研究從整體、細(xì)胞、分子三水平上,確定鼠疫菌yscW突變株及互補(bǔ)株對(duì)原代小鼠巨噬細(xì)胞及RAW264.7細(xì)胞系生物學(xué)功能的影響,并對(duì)YscW蛋白本身的巨噬細(xì)胞免疫應(yīng)答狀態(tài)做出綜合評(píng)價(jià),特別是感染后或過(guò)表達(dá)后巨噬細(xì)胞的吞噬能力以及抗原提呈能力進(jìn)行系統(tǒng)研究。在前期實(shí)驗(yàn)篩選到鼠疫菌YscW與巨噬細(xì)胞Rnf149可能存在相互作用的基礎(chǔ)上,進(jìn)一步驗(yàn)證兩者相互作用,以期探究鼠疫菌III型分泌系統(tǒng)結(jié)構(gòu)蛋白YscW的致病機(jī)制,對(duì)鼠疫菌結(jié)構(gòu)蛋白的新功能研究具有重要的意義。
[Abstract]:Yersinia pestis is a severe infectious disease caused by Yersinia pestis. The epidemic of Yersinia pestis has brought great disaster to human beings. Therefore, the study on the pathogenic mechanism of Yersinia pestis is an important breakthrough point for the prevention and control of plague. The key link is to survive and reproduce in macrophages. The bacterium interacts with the macrophages not only to survive, but also to synthesize a large number of virulence factors. Eventually the macrophages break down, releasing Yersinia pestis with strong anti-phagocytosis ability, and to prepare for the secretion of virulence factors. One of the virulence of Yersinia pestis is the type III secretion system (T3SS) encoded by pCD1 plasmid, which is transfected into eukaryotic cells via six effector proteins to interrupt the host defense mechanism. In this study, the structure of mice injected with Yersinia pestis T3SS was studied. YscW protein was used as the research object to analyze the effect of yscW gene deletion on the virulence of Yersinia pestis; to explore the macrophage effect of yscW mutant and complementary strains; to study whether YscW protein directly affects the survival and immune regulation of macrophages; and to explore the possible mechanism of YscW protein function.
Firstly, yscW mutant was constructed by suicide plasmid method, and then a complementary strain was constructed by introducing the recombinant plasmid pACYC184-YscW which was close to the copy number of Y. pestis. The effect of yscW on the pathogenicity of Y. pestis was observed by LD50 assay. The results showed that Y. pestis lacked yscW gene and its pathogenicity was significantly weakened. Observation indicates that YscW affects macrophage morphology.
Then the mouse abdominal infection model was constructed to analyze the effect of wild Yersinia pestis strain, yscW mutant and complementary strain on the survival of macrophages. In vivo, in vitro and in vivo experiments showed that yscW mutant significantly reduced the apoptosis of macrophages, while complementary strain recovered the apoptotic changes caused by the mutant. In addition, cell cycle analysis showed that there was no significant difference among the three strains. Meanwhile, the expression of Caspase-3 was consistent with the changes of cell apoptosis at the cellular level after infection. However, after overexpression of YscW to RAW264.7 cells, there was no significant difference in cell apoptosis and cell cycle between the experimental group and the control group, suggesting that YscW was not a direct effector protein inducing cell apoptosis. YopJ was the only one of the six effector proteins in the secretory system of Yersinia pestis type III related to cell apoptosis. The results showed that YscW mutation resulted in the decrease of YopJ secretion. Therefore, the decrease of YopJ secretion of Y. pestis YscW mutant was one of the mechanisms leading to the decrease of YopJ secretion.
The function changes of macrophages infected by different strains of Y. pestis were studied. The results showed that Y. pestis YscW affected the immune regulation function of macrophages. Phagocytosis, antigen presenting ability and cytokine secretion were the three main functions of macrophages. The phagocytic function of macrophages in wild strains, mutants and complementary strains was significantly lower than that in normal macrophages, indicating that the phagocytic function of host macrophages decreased after infection by Yersinia pestis, and the phagocytic function of cells infected with Y. pestis yscW mutant was lower than that of wild strains and complementary strains. The phagocytic function of YscW gene was also significantly enhanced, indicating that yscW gene directly or indirectly participated in the phagocytic response of host cells regulated by pathogenic bacteria. The results showed that the antigen presenting ability of macrophages infected by the mutant strain was significantly higher than that of the cells infected by the wild strain under the same conditions, while the antigen presenting ability of the cells overexpressing YscW was significantly lower than that of the control group. These results indicated that YscW was direct. In addition, the detection of macrophages and over-expressed cell surface molecules after infection further confirmed the effect of YscW on cell antigen presenting ability. TNF-a secretion of macrophages after infection was significantly higher than that of the other two strains, suggesting that yscW gene-deleted macrophages increased the release of pro-inflammatory factors. The effect of YscW on the secretion of cytokines was studied from the point of view of influencing the secretion of cytokines. The regulation of reaction interferes with the normal physiological functions of cells.
Previous work in our laboratory has found that Y. pestis YscW protein may interact with macrophage Rnf149 protein, which indicates whether the biological effects of YscW macrophages observed above are related to the interaction between YscW and Rnf149. Therefore, the validation of the interaction between the two proteins is the basis for subsequent experiments. The protein was purified by nickel column and GS-4B column. The binding of YscW to macrophage target protein Rnf149 was confirmed by GST-Pull Down assay in vitro.
In this study, we determined the effects of Y. pestis yscW mutants and complementary strains on the biological functions of primary mouse macrophages and RAW264.7 cell lines from the overall, cellular and molecular levels, and made a comprehensive evaluation of the macrophage immune response status of YscW protein itself, especially the phagocytosis ability of macrophages after infection or overexpression. The antigen presenting ability of Y. pestis YscW was systematically studied. On the basis of the possible interaction between Y. pestis YscW and macrophage Rnf149, the interaction between Y. pestis YscW and macrophage Rnf149 was further verified in order to explore the pathogenic mechanism of Y. pestis type III secretory system structural protein YscW. Righteousness.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R378

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 江凌曉;江凌云;;S系統(tǒng)及時(shí)序布爾網(wǎng)絡(luò)模型構(gòu)建鼠疫耶爾森菌基因調(diào)控網(wǎng)絡(luò)的初步研究[J];熱帶醫(yī)學(xué)雜志;2011年09期

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本文編號(hào)：2207827