结核分枝杆菌Mce2E抑制宿主固有免疫并促进肿瘤细胞增殖

发布时间：2018-01-01 22:16

  本文关键词：结核分枝杆菌Mce2E抑制宿主固有免疫并促进肿瘤细胞增殖　出处：《安徽大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 结核分枝杆菌 Mce2E ERK eEF1A1 细胞增殖

【摘要】：结核分枝杆菌是一种兼性胞内致病菌,以巨噬细胞为主要的宿主细胞,并在其中存活和生长。哺乳动物细胞入侵蛋白(mammalian cell entry,Mce)Mce是由mce基因编码的一组分泌或者膜表面粘附蛋白,已被证明具有促进分枝杆菌入侵宿主细胞的功能。其中mce2操纵子就与分枝杆菌的毒力和肉芽肿形成有关,但是,mce2操纵子编码的Mce2E蛋白的相关功能尚不清楚。脂多糖以及病原菌的感染会刺激巨噬细胞中核因子K轻链B细胞活化因子(nuclear factor kappa-light-chain-enchancer of activated B cell,NF-κB)和丝裂原活化的蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路,进而促进TNFα和IL-1β等炎症因子的释放,激活宿主固有免疫应答。结核分枝杆菌表面的LAM具有类似脂多糖的功能。因此,我们在HEK293T细胞中瞬时表达Mce2E蛋白,通过双荧光报告基因检测对NF-κB和MAPKs信号通路的影响。实验结果表明,Mce2E蛋白对TNFα激活的NF-κB信号通路没有明显的影响。但是能部分地抑制RacL61诱导的JNK/p38信号通路的激活。而对于RasV12、v-Raf、MEK1-ED 诱导的 ERK(Extracellular regulated protein kinase)信号通路的活化有明显的抑制作用。于是,我们针对ERK信号通路做了相应的研究,按照Ras-Raf-MEK-ERK通路,我们推测Mce2E蛋白可能抑制节点在MEK1或ERK1/2上。随后,我们通过免疫共沉淀实验确定了 Mce2E和ERK1/2的相互作用,而不是MEK1。在蛋白结构模序的预测中,我们发现Mce2E蛋白具有一种类似于真核细胞中特异地结合ERK1/2的模序,即D模序。我们构建了 D模序的突变体Mce2E(AAA),研究表明Mce2E-AAA失去抑制ERK活化的功能。由此可见Mce2E蛋白主要通过D模序影响ERK信号通路的活化。此外,我们在 CFU(Colony-Forming Units)、QRT-PCR(Quantitative Real-time PCR)、ELISA(Enzyme Linked Immunosorbent Assay)等相关实验发现,Mce2E能促进结核分枝杆菌在巨噬细胞中的存活,并且能下调TNFα和IL-6炎症因子的表达,从而达到促进分枝杆菌胞内存活的目的。结核分枝杆菌不仅能进入巨噬细胞,还能入侵肺上皮细胞等一些非专职吞噬细胞。我们在转染Mce2E至上皮细胞中时注意到转染了 Mce2E的细胞的增殖能力明显增强,这个现象提示Mce2E可能调控肿瘤细胞的增殖并因此而促进肿瘤的发展进程。在酵母双杂交实验中,我们发现筛选到的Mce2E互作蛋白中,许多与细胞增殖及迁移相关。相关文献也提示Mce家族蛋白确实具有促增殖,迁移的作用。于是我们用过表达Mce2E和沉默Mce2E的菌株分别去感染A549细胞,结果发现Mce2E能促进A549细胞的增殖。与此同时,我们还研究了 Mce2E对A549细胞迁移、侵袭的影响,实验结果表明,Mce2E蛋白对A549细胞的迁移和侵袭能力同样具有促进作用。随后的裸鼠实验进一步证明,过表达Mce2E菌株感染的肿瘤体积明显比没有过表达Mce2E的大,沉默Mce2E的菌株的肿瘤体积比野生型菌株感染的小。在酵母双杂交的实验中,我们筛选到与增殖相关的蛋白eEF1A1。过表达Mce2E的菌株感染实验中发现CHX处理的情况下,eEF1A1蛋白并没有随着时间延长而降解,表明过表达Mce2E菌株能稳定eEF1A1蛋白。随后,通过体内泛素化实验表明Mce2E蛋白能减弱对eEF1A1的泛素化修饰,在分别共转HA-K48(only),HA-K63(only)质粒的体外泛素化实验中(除了 48或63位以外所有的赖氨酸都突变为精氨酸的泛素突变体),我们发现Mce2E蛋白很明显地阻断K48对eEF1A1的泛素化修饰作用,而K63泛素链则不明显。该结果说明Mce2E阻断的是eEF1A1蛋白K48位的泛素化修饰。综上所述,我们的研究结果揭示了结核分枝杆菌Mce2E蛋白抑制宿主固有免疫和促进分枝杆菌胞内存活的分子机制;并进一步发现Mce2E蛋白可以促进肿瘤细胞A549的增殖、迁移以及侵袭能力。该成果为炎癌转化相关研究提供了一个新的视角,以便更好地攻克慢性感染和炎症所致的肿瘤这一世界难题。
[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.

【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378.911

【参考文献】

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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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