本文關(guān)鍵詞：結(jié)核分枝桿菌Mce2E抑制宿主固有免疫并促進(jìn)腫瘤細(xì)胞增殖　出處：《安徽大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

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【摘要】：結(jié)核分枝桿菌是一種兼性胞內(nèi)致病菌,以巨噬細(xì)胞為主要的宿主細(xì)胞,并在其中存活和生長(zhǎng)。哺乳動(dòng)物細(xì)胞入侵蛋白(mammalian cell entry,Mce)Mce是由mce基因編碼的一組分泌或者膜表面粘附蛋白,已被證明具有促進(jìn)分枝桿菌入侵宿主細(xì)胞的功能。其中mce2操縱子就與分枝桿菌的毒力和肉芽腫形成有關(guān),但是,mce2操縱子編碼的Mce2E蛋白的相關(guān)功能尚不清楚。脂多糖以及病原菌的感染會(huì)刺激巨噬細(xì)胞中核因子K輕鏈B細(xì)胞活化因子(nuclear factor kappa-light-chain-enchancer of activated B cell,NF-κB)和絲裂原活化的蛋白激酶(mitogen-activated protein kinase,MAPK)信號(hào)通路,進(jìn)而促進(jìn)TNFα和IL-1β等炎癥因子的釋放,激活宿主固有免疫應(yīng)答。結(jié)核分枝桿菌表面的LAM具有類似脂多糖的功能。因此,我們?cè)贖EK293T細(xì)胞中瞬時(shí)表達(dá)Mce2E蛋白,通過雙熒光報(bào)告基因檢測(cè)對(duì)NF-κB和MAPKs信號(hào)通路的影響。實(shí)驗(yàn)結(jié)果表明,Mce2E蛋白對(duì)TNFα激活的NF-κB信號(hào)通路沒有明顯的影響。但是能部分地抑制RacL61誘導(dǎo)的JNK/p38信號(hào)通路的激活。而對(duì)于RasV12、v-Raf、MEK1-ED 誘導(dǎo)的 ERK(Extracellular regulated protein kinase)信號(hào)通路的活化有明顯的抑制作用。于是,我們針對(duì)ERK信號(hào)通路做了相應(yīng)的研究,按照Ras-Raf-MEK-ERK通路,我們推測(cè)Mce2E蛋白可能抑制節(jié)點(diǎn)在MEK1或ERK1/2上。隨后,我們通過免疫共沉淀實(shí)驗(yàn)確定了 Mce2E和ERK1/2的相互作用,而不是MEK1。在蛋白結(jié)構(gòu)模序的預(yù)測(cè)中,我們發(fā)現(xiàn)Mce2E蛋白具有一種類似于真核細(xì)胞中特異地結(jié)合ERK1/2的模序,即D模序。我們構(gòu)建了 D模序的突變體Mce2E(AAA),研究表明Mce2E-AAA失去抑制ERK活化的功能。由此可見Mce2E蛋白主要通過D模序影響ERK信號(hào)通路的活化。此外,我們?cè)?CFU(Colony-Forming Units)、QRT-PCR(Quantitative Real-time PCR)、ELISA(Enzyme Linked Immunosorbent Assay)等相關(guān)實(shí)驗(yàn)發(fā)現(xiàn),Mce2E能促進(jìn)結(jié)核分枝桿菌在巨噬細(xì)胞中的存活,并且能下調(diào)TNFα和IL-6炎癥因子的表達(dá),從而達(dá)到促進(jìn)分枝桿菌胞內(nèi)存活的目的。結(jié)核分枝桿菌不僅能進(jìn)入巨噬細(xì)胞,還能入侵肺上皮細(xì)胞等一些非專職吞噬細(xì)胞。我們?cè)谵D(zhuǎn)染Mce2E至上皮細(xì)胞中時(shí)注意到轉(zhuǎn)染了 Mce2E的細(xì)胞的增殖能力明顯增強(qiáng),這個(gè)現(xiàn)象提示Mce2E可能調(diào)控腫瘤細(xì)胞的增殖并因此而促進(jìn)腫瘤的發(fā)展進(jìn)程。在酵母雙雜交實(shí)驗(yàn)中,我們發(fā)現(xiàn)篩選到的Mce2E互作蛋白中,許多與細(xì)胞增殖及遷移相關(guān)。相關(guān)文獻(xiàn)也提示Mce家族蛋白確實(shí)具有促增殖,遷移的作用。于是我們用過表達(dá)Mce2E和沉默Mce2E的菌株分別去感染A549細(xì)胞,結(jié)果發(fā)現(xiàn)Mce2E能促進(jìn)A549細(xì)胞的增殖。與此同時(shí),我們還研究了 Mce2E對(duì)A549細(xì)胞遷移、侵襲的影響,實(shí)驗(yàn)結(jié)果表明,Mce2E蛋白對(duì)A549細(xì)胞的遷移和侵襲能力同樣具有促進(jìn)作用。隨后的裸鼠實(shí)驗(yàn)進(jìn)一步證明,過表達(dá)Mce2E菌株感染的腫瘤體積明顯比沒有過表達(dá)Mce2E的大,沉默Mce2E的菌株的腫瘤體積比野生型菌株感染的小。在酵母雙雜交的實(shí)驗(yàn)中,我們篩選到與增殖相關(guān)的蛋白eEF1A1。過表達(dá)Mce2E的菌株感染實(shí)驗(yàn)中發(fā)現(xiàn)CHX處理的情況下,eEF1A1蛋白并沒有隨著時(shí)間延長(zhǎng)而降解,表明過表達(dá)Mce2E菌株能穩(wěn)定eEF1A1蛋白。隨后,通過體內(nèi)泛素化實(shí)驗(yàn)表明Mce2E蛋白能減弱對(duì)eEF1A1的泛素化修飾,在分別共轉(zhuǎn)HA-K48(only),HA-K63(only)質(zhì)粒的體外泛素化實(shí)驗(yàn)中(除了 48或63位以外所有的賴氨酸都突變?yōu)榫彼岬姆核赝蛔凅w),我們發(fā)現(xiàn)Mce2E蛋白很明顯地阻斷K48對(duì)eEF1A1的泛素化修飾作用,而K63泛素鏈則不明顯。該結(jié)果說明Mce2E阻斷的是eEF1A1蛋白K48位的泛素化修飾。綜上所述,我們的研究結(jié)果揭示了結(jié)核分枝桿菌Mce2E蛋白抑制宿主固有免疫和促進(jìn)分枝桿菌胞內(nèi)存活的分子機(jī)制;并進(jìn)一步發(fā)現(xiàn)Mce2E蛋白可以促進(jìn)腫瘤細(xì)胞A549的增殖、遷移以及侵襲能力。該成果為炎癌轉(zhuǎn)化相關(guān)研究提供了一個(gè)新的視角,以便更好地攻克慢性感染和炎癥所致的腫瘤這一世界難題。
[Abstract]:Mycobacterium tuberculosis is a facultative intracellular pathogen in macrophages as the host cells, and the survival and growth of mammalian cells. The invasion protein (mammalian cell entry, Mce Mce) is a MCE gene encoding a group of secretory or membrane surface adhesion protein, has been shown to promote invasion of host Mycobacterium the function of cells. The mce2 operon with mycobacterial virulence and granuloma formation, but the related function of mce2 operon encoding the Mce2E protein is not clear. And the infection of pathogenic bacteria lipopolysaccharide stimulates macrophage nuclear factor K light chain B cell activating factor (nuclear factor kappa-light-chain-enchancer of activated B cell, NF- K B) and mitogen activated protein kinase (mitogen-activated protein, kinase, MAPK) signaling pathway, thereby promoting TNF alpha and IL-1 beta inflammatory factor release, activation The innate immune response of the host. The surface of Mycobacterium tuberculosis LAM with similar LPS function. Therefore, the transient expression of Mce2E protein in HEK293T cells, detected by double fluorescent reporter gene of NF- kappa B and MAPKs signaling pathway. The experimental results show that the NF- B signaling pathway and activation of Mce2E protein of TNF alpha is not obvious the influence. But can activate partially inhibits JNK/p38 signaling pathway induced by RacL61. But for RasV12, v-Raf, MEK1-ED induced ERK (Extracellular regulated protein kinase) signaling pathway activation has obvious inhibitory effects. So, we in the ERK signaling pathway was investigated, according to the Ras-Raf-MEK-ERK pathway, we hypothesized that Mce2E protein may inhibit the node in MEK1 or ERK1/2. Then, we through co immunoprecipitation experiments to determine the interaction between Mce2E and ERK1/2, but not MEK1. in protein In order to predict model, we found that Mce2E protein has a similar to eukaryotic cell specific ERK1/2 binding motif, D motif. We constructed a D motif mutant Mce2E (AAA), the research shows that Mce2E-AAA lost ERK activation function. Thus the Mce2E protein mainly through the activation effect of D motif of the ERK signaling pathway. In addition, we in the CFU (Colony-Forming Units), QRT-PCR (Quantitative Real-time PCR), ELISA (Enzyme Linked Immunosorbent Assay) found that Mce2E can promote the related experiments of Mycobacterium tuberculosis in macrophages in survival, expression and down-regulation of TNF alpha and IL-6 and inflammatory factors. To promote the intracellular survival of Mycobacterium tuberculosis. The purpose is not only able to enter macrophages, but also invasion of lung epithelial cells and some other non professional phagocytes. Note that we first in transfected Mce2E cells in the skin The transfection of Mce2E cells significantly enhanced the proliferation of this phenomenon, suggesting that Mce2E may regulate tumor cell proliferation and promote tumor development process. In the yeast two hybrid experiments, we found that the screening of the Mce2E protein interaction, many associated with cell proliferation and migration. The related literature also revealed that the Mce protein family it can promote the proliferation and migration. So we used the expression of Mce2E and Mce2E strains were silent to infect A549 cells. The results showed that Mce2E could promote the proliferation of A549 cells. At the same time, we also studied the effect of Mce2E on A549 cell migration, invasion effect, experimental results show that Mce2E protein on A549 cell migration and the invasion ability also has a role in promoting. Then nude mice experiment further proved that overexpression of Mce2E strain infected tumor volume was not over expressed Mce2E, silent Mce2E The strains of the tumor volume ratio of infection of wild-type strains. In yeast two hybrid experiments, we screened and proliferation related protein eEF1A1. expression Mce2E strain infection experiments showed that the CHX, eEF1A1 protein and no degradation over time, showed that over expression of Mce2E strains can stabilize eEF1A1 protein then, through in vivo ubiquitination assays showed that Mce2E protein can weaken the ubiquitination of eEF1A1, respectively in the co transfection of HA-K48 (only), HA-K63 (only) in vitro ubiquitination experiments in plasmid (except for the 48 or 63 lysines outside all mutant ubiquitin mutants of arginine), we found Mce2E protein obviously blocked the ubiquitination effect of K48 on eEF1A1, K63 and ubiquitin chain is not obvious. The results indicate that Mce2E is blocking the ubiquitination of the eEF1A1 protein K48. In summary, the results of our study Reveals the Mce2E protein of Mycobacterium tuberculosis and Mycobacterium inhibitory molecular mechanisms promoting intracellular survival of host innate immunity; and further found that Mce2E protein can promote A549 cell proliferation, migration and invasion ability. The results provide a new perspective for inflammatory cancer transformation related research, in order to better overcome the chronic infection and inflammation tumors due to problems of the world.

【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R378.911

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Ameer Khusro;Chirom Aarti;Paul Agastian;;Anti-tubercular peptides:A quest of future therapeutic weapon to combat tuberculosis[J];Asian Pacific Journal of Tropical Medicine;2016年11期

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本文編號(hào)：1366444