本文選題：小窩　切入點：小窩蛋白-1　出處：《華中農(nóng)業(yè)大學》2017年碩士論文

【摘要】：腸外致病性大腸桿菌(Ex PEC)是一種人獸共患病原菌,根據(jù)報道表明,其感染范圍遠遠大于其他已知的大腸桿菌,所以造成巨大損失。臨床數(shù)據(jù)統(tǒng)計表明,ExPEC是一種非常重要致中樞神經(jīng)系統(tǒng)(CNS)感染病原菌。中樞神經(jīng)系統(tǒng)(CNS)是機體中密封的組織器官。通常病原微生物感染CNS的方式,主要是通過兩種途徑:第一種是通過感染鄰近組織繼而向顱內(nèi)擴散,第二種是通過血源感染,后續(xù)穿透血腦屏障造成CNS感染。其中,病原微生物穿過血腦屏障造成感染是最常見的一種形式。腦膜炎是最常見的一種CNS感染,病原微生物通過粘附血管內(nèi)皮細胞進行刺激,并且促使中性白細胞進入CNS,從而來引發(fā)炎性進程。內(nèi)皮細胞表面存在豐富的小窩(caveolae),這是一種呈凹陷狀的細胞膜表面結構,在信號傳導和內(nèi)吞中都發(fā)揮巨大作用。其主要成分是小窩蛋白(caveolins)和膽固醇。膽固醇是小窩結構的含量最高的組成分,小窩蛋白-1(caveolin-1)是內(nèi)皮細胞中小窩最主要的功能蛋白,并且這兩種組分對于小窩結構和功能的維持都是必要的。通過一系列體外實驗,結果發(fā)現(xiàn)caveolin-1與不飽和長鏈脂肪酸具有高度親和力,這也表明caveolin-1可能參與小窩中膽固醇的運輸。然而,膽固醇和caveolin-1在ExPEC突破宿主血腦屏障,繼而引發(fā)機體CNS感染中的作用并不清楚。本實驗通過豬源腸外致病性大腸桿菌菌株PCN033作為研究對象,采用HBMECs模擬血腦屏障(BBB)體外模型,通過檢測caveolin-1在感染前后的轉(zhuǎn)錄和翻譯變化水平和相關信號通路驗證,結果表明,caveolae在PCN033入侵中具有重要作用。這也為研究新的抗ExPEC感染提供新的靶點和理論基礎。取得結果如下:1.Caveolae存在于腦微血管內(nèi)皮細胞(BMEC)表面Caveolin-1是caveolae的標志性蛋白,本實驗通過提取HBMEC細胞的RNA并反轉(zhuǎn)錄,采用PCR檢測感染前后的caveolin-1、caveolin-2、caveolin-3基因,檢測結果顯示HBMEC中轉(zhuǎn)錄caveolin-1和caveolin-2。同時提取不同感染時間的HBMEC細胞總蛋白,檢測caveolin-1和caveolin-2蛋白變化及磷酸化。結果顯示:在HBMEC中存在caveolin中caveolin-1和caveolin-2兩種形式,并且,在細胞受到感染后主要通過caveolin-1的磷酸化發(fā)揮功能。2.Caveolae在內(nèi)皮功能中的作用內(nèi)皮細胞表面存在豐富的caveolae結構。本研究通過研究在血源性細菌感染中,使用caveolae的抑制劑是否會影響ExPEC侵入腦組織能力變化,和干擾以及過表達caveolin-1后ExPEC入侵HBMEC能力變化,從而探討caveolae在其中的作用。結果表明:在使用caveolae抑制劑之后,ExPEC入侵腦組織能力相應也有下降。同時,干擾caveolin-1后,Ex PEC入侵能力顯著降低;過表達caveolin-1后,Ex PEC入侵能力顯著升高。這說明,細菌能夠通過caveolae介導的內(nèi)吞穿過血管內(nèi)皮細胞從而導致腦膜炎,并且caveolin-1在其中起重要作用。3.Caveolin的調(diào)節(jié)信號傳導分子機制SREBP是膽固醇敏感器,在胞內(nèi)膽固醇含量發(fā)生變化時會進行負反饋調(diào)節(jié),通過裂解之后入核與SRE元件結合從而負反饋調(diào)節(jié)基因轉(zhuǎn)錄水平。但是對于SREBP在本實驗中的作用形式仍是未知。本研究通過采用RT-PCR的方法檢測HBMEC中SREBP三種形態(tài)轉(zhuǎn)錄水平變化,來探究其作用形式。結果表明:發(fā)揮主要功能的是SREBP-1a和SREBP-1c。并且在抑制SREBP激活之后,CAV-1的轉(zhuǎn)錄水平也有了極其顯著的提高。
[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.

【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.61

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