Caveolae介导肠外致病性大肠杆菌侵入脑微血管内皮细胞的机制研究

发布时间：2018-04-03 14:09

本文选题：小窝　切入点：小窝蛋白-1　出处：《华中农业大学》2017年硕士论文

【摘要】：肠外致病性大肠杆菌(Ex PEC)是一种人兽共患病原菌,根据报道表明,其感染范围远远大于其他已知的大肠杆菌,所以造成巨大损失。临床数据统计表明,ExPEC是一种非常重要致中枢神经系统(CNS)感染病原菌。中枢神经系统(CNS)是机体中密封的组织器官。通常病原微生物感染CNS的方式,主要是通过两种途径:第一种是通过感染邻近组织继而向颅内扩散,第二种是通过血源感染,后续穿透血脑屏障造成CNS感染。其中,病原微生物穿过血脑屏障造成感染是最常见的一种形式。脑膜炎是最常见的一种CNS感染,病原微生物通过粘附血管内皮细胞进行刺激,并且促使中性白细胞进入CNS,从而来引发炎性进程。内皮细胞表面存在丰富的小窝(caveolae),这是一种呈凹陷状的细胞膜表面结构,在信号传导和内吞中都发挥巨大作用。其主要成分是小窝蛋白(caveolins)和胆固醇。胆固醇是小窝结构的含量最高的组成分,小窝蛋白-1(caveolin-1)是内皮细胞中小窝最主要的功能蛋白,并且这两种组分对于小窝结构和功能的维持都是必要的。通过一系列体外实验,结果发现caveolin-1与不饱和长链脂肪酸具有高度亲和力,这也表明caveolin-1可能参与小窝中胆固醇的运输。然而,胆固醇和caveolin-1在ExPEC突破宿主血脑屏障,继而引发机体CNS感染中的作用并不清楚。本实验通过猪源肠外致病性大肠杆菌菌株PCN033作为研究对象,采用HBMECs模拟血脑屏障(BBB)体外模型,通过检测caveolin-1在感染前后的转录和翻译变化水平和相关信号通路验证,结果表明,caveolae在PCN033入侵中具有重要作用。这也为研究新的抗ExPEC感染提供新的靶点和理论基础。取得结果如下:1.Caveolae存在于脑微血管内皮细胞(BMEC)表面Caveolin-1是caveolae的标志性蛋白,本实验通过提取HBMEC细胞的RNA并反转录,采用PCR检测感染前后的caveolin-1、caveolin-2、caveolin-3基因,检测结果显示HBMEC中转录caveolin-1和caveolin-2。同时提取不同感染时间的HBMEC细胞总蛋白,检测caveolin-1和caveolin-2蛋白变化及磷酸化。结果显示:在HBMEC中存在caveolin中caveolin-1和caveolin-2两种形式,并且,在细胞受到感染后主要通过caveolin-1的磷酸化发挥功能。2.Caveolae在内皮功能中的作用内皮细胞表面存在丰富的caveolae结构。本研究通过研究在血源性细菌感染中,使用caveolae的抑制剂是否会影响ExPEC侵入脑组织能力变化,和干扰以及过表达caveolin-1后ExPEC入侵HBMEC能力变化,从而探讨caveolae在其中的作用。结果表明:在使用caveolae抑制剂之后,ExPEC入侵脑组织能力相应也有下降。同时,干扰caveolin-1后,Ex PEC入侵能力显著降低;过表达caveolin-1后,Ex PEC入侵能力显著升高。这说明,细菌能够通过caveolae介导的内吞穿过血管内皮细胞从而导致脑膜炎,并且caveolin-1在其中起重要作用。3.Caveolin的调节信号传导分子机制SREBP是胆固醇敏感器,在胞内胆固醇含量发生变化时会进行负反馈调节,通过裂解之后入核与SRE元件结合从而负反馈调节基因转录水平。但是对于SREBP在本实验中的作用形式仍是未知。本研究通过采用RT-PCR的方法检测HBMEC中SREBP三种形态转录水平变化,来探究其作用形式。结果表明:发挥主要功能的是SREBP-1a和SREBP-1c。并且在抑制SREBP激活之后,CAV-1的转录水平也有了极其显著的提高。
[Abstract]:Extraintestinal pathogenic Escherichia coli (Ex PEC) is a zoonotic pathogen, according to the report shows that the infection range is far greater than other known Escherichia coli, so that caused huge losses. Clinical data, ExPEC is a very important cause of central nervous system (CNS) infection pathogens. The central nervous system (CNS) is sealed in the body tissues and organs. Usually the infection of pathogenic microorganisms CNS, mainly through two ways: the first one is the infection of adjacent tissue and then to intracranial spread through the second blood borne infection, subsequent penetrating blood brain barrier caused by CNS infection. Among them, across the blood-brain barrier caused by pathogenic microorganisms infection is the most common form of meningitis is a most common CNS infection, pathogenic microorganisms were stimulated by vascular endothelial cell adhesion, and promote neutrophil cells into CNS, so as to cause inflammation Process. There are abundant caveolin endothelial cell surface (caveolae), which is a cell membrane sunken surface structure, can play a significant role in signal transduction and endocytosis. It is the main component of caveolin-1 (Caveolins) and cholesterol. Cholesterol is the highest content of caveolae structure composition, caveolin -1 (caveolin-1) is the main protein function of caveolae in endothelial cells, and these two components to maintain the structure and function of caveolae are necessary. Through a series of in vitro experiments, the results showed that caveolin-1 and unsaturated long-chain fatty acids with high affinity, which suggests that caveolin-1 may be involved in caveolae cholesterol transport. However, cholesterol and caveolin-1 in host ExPEC to break the blood-brain barrier, which led to the role of CNS infection is not clear. The experiments of swine extraintestinal pathogenic Escherichia coli strains PCN033 As the research object, using HBMECs to simulate the blood brain barrier (BBB) in vitro model, through the detection of caveolin-1 in transcription and translation levels and related signaling pathways verified before and after infection. The results show that caveolae plays an important role in the invasion of PCN033. This is a new research to provide a new target and theoretical basis of anti ExPEC infection. The results are as follows: 1.Caveolae exists in brain microvascular endothelial cells (BMEC) surface Caveolin-1 is the marker protein of caveolae, through the experiments of extraction of HBMEC cell RNA and reverse transcription, by using PCR before and after infection of caveolin-1, Caveolin-2, caveolin-3 gene, and test results showed that the transcription of caveolin-1 and caveolin-2. HBMEC in different extraction time of infection total protein of HBMEC cells, the detection of caveolin-1 and Caveolin-2 protein and phosphorylation. The results showed that the presence of caveolin-1 and Caveolin-2 caveolin in HBMEC Two kinds of forms, and in the cells after the infection are rich caveolae structure function of.2.Caveolae on endothelial function of endothelial cell surface through the phosphorylation of caveolin-1. Through the study on blood borne bacterial infection, inhibition of caveolae will affect the ExPEC invasion changes of brain tissue, and interference and after overexpression of caveolin-1 ExPEC HBMEC invasion ability changes, so as to explore the role of caveolae. The results showed that: after using caveolae inhibitor, ExPEC invasion of brain tissue capacity also decreased. At the same time, caveolin-1 after Ex PEC interference, the invasion ability decreased significantly; after overexpression of caveolin-1, Ex PEC were significantly increased. The invasion ability endocytosis, bacteria can be mediated by caveolae through endothelial cells leading to meningitis, caveolin-1 and.3.Ca played an important role in the SREBP signal transduction molecular mechanism regulating veolin cholesterol sensor, the intracellular cholesterol content changes will be negatively regulated by cleavage after nuclear and SRE binding to the negative feedback regulation of gene transcription. But for SREBP in this experiment with the form remains unknown. In this study, through the use of SREBP RT-PCR methods to detect HBMEC in three forms of transcription level, to explore its role in the form of play. The results show that the main function of the SREBP-1a and SREBP-1c. and after inhibition of SREBP activation, the transcription level of CAV-1 also has a very significant improvement.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

【参考文献】