【摘要】：研究背景心血管疾病是全世界居首位的死亡原因,其最主要的病因是動(dòng)脈粥樣硬化。動(dòng)脈粥樣硬化的本質(zhì)是由多種因素引起的血管慢性炎癥,其中病原體的多重感染,即“感染負(fù)荷(infectious burden) ",被認(rèn)為是動(dòng)脈粥樣硬化新的獨(dú)立危險(xiǎn)因素。感染因素可通過(guò)直接作用、觸發(fā)全身性炎癥和啟動(dòng)自身免疫反應(yīng)等多種方式對(duì)動(dòng)脈壁造成損害,導(dǎo)致動(dòng)脈粥樣硬化的發(fā)生發(fā)展,其中病原體激活血小板所導(dǎo)致的血管炎癥和血栓形成具有重要作用。金黃色葡萄球菌(Staphylococcus aureus, S. aureus)是臨床常見(jiàn)的致病菌之一。有研究顯示,血行感染S. aureus會(huì)增加急性心肌梗死和腦卒中的風(fēng)險(xiǎn),但機(jī)制尚不明確,目前認(rèn)為可能與血小板激活、炎癥反應(yīng)增強(qiáng)以及血液的高凝狀態(tài)有關(guān)。本課題小組前期研究發(fā)現(xiàn),金黃色葡萄球菌超抗原樣蛋白-5 (Staphylococcal superantigen-like protein 5, SSL5)可以激活血小板,一次性靜脈注射SSL5還可導(dǎo)致小鼠肺動(dòng)脈內(nèi)形成血栓。血小板不僅在血栓和止血的過(guò)程中發(fā)揮作用,同時(shí)還是重要的免疫和炎癥細(xì)胞,其可以通過(guò)激活產(chǎn)生的微粒或與白細(xì)胞相互作用導(dǎo)致炎癥反應(yīng)。因此,我們推測(cè)SSL5可能通過(guò)激活血小板而誘發(fā)炎癥反應(yīng),探討其機(jī)制可以為闡明感染負(fù)荷在動(dòng)脈粥樣硬化中的作用提供新的實(shí)驗(yàn)證據(jù)。研究?jī)?nèi)容1.采用流式細(xì)胞術(shù),鑒定重組SSL5的生物學(xué)活性,檢測(cè)外周血中PMPs數(shù)量以及SSL5激活的PMPs(SSL5-PMPs)表面分子的特征；利用掃描電鏡觀察SSL5-PMPs的形態(tài)學(xué)特征；體外制備SSL5-PMPs以及ADP激活的PMPs (ADP-PMPs)和凋亡產(chǎn)生的PMPs (ap-PMPs),作為后續(xù)實(shí)驗(yàn)的刺激物和對(duì)照。2.以人外周血單核細(xì)胞及THP-1細(xì)胞作為實(shí)驗(yàn)對(duì)象,利用CCK-8法檢測(cè)SSL5對(duì)單核細(xì)胞活力的影響；掃描電鏡觀察SSL5-PMPs與單核細(xì)胞的結(jié)合,流式細(xì)胞術(shù)進(jìn)一步檢測(cè)兩者結(jié)合的時(shí)效關(guān)系和差異性；以產(chǎn)生炎癥介質(zhì)(細(xì)胞因子、趨化因子和基質(zhì)金屬蛋白酶)及發(fā)生遷移作為單核細(xì)胞炎癥效應(yīng)的指標(biāo),實(shí)時(shí)定量PCR檢測(cè)炎癥介質(zhì)mRNA的表達(dá),ELISA法檢測(cè)細(xì)胞因子及趨化因子釋放的數(shù)量,明膠酶譜法檢測(cè)蛋白酶的活性,transwell遷移試驗(yàn)檢測(cè)細(xì)胞的遷移。同時(shí)一并觀察對(duì)照PMPs (ADP-PMPs及ap-PMPs)和SSL5對(duì)單核細(xì)胞炎癥介質(zhì)mRNA表達(dá)的影響,以探討PMPs致單核細(xì)胞炎癥效應(yīng)的差異性及SSL5可能的病理作用。3.以THP-1細(xì)胞作為實(shí)驗(yàn)對(duì)象,利用中和性抗體干擾SSL5-PMPs與THP-1細(xì)胞間CD40L/CD40和P-選擇素/PSGL-1的結(jié)合,觀察其對(duì)THP-1細(xì)胞產(chǎn)生細(xì)胞因子及遷移的影響,以明確介導(dǎo)SSL5-PMPs與THP-1細(xì)胞發(fā)生相互作用的分子基礎(chǔ)；利用siRNA沉默THP-1細(xì)胞CD40或TRAF6的基因表達(dá),觀察其對(duì)THP-1細(xì)胞產(chǎn)生細(xì)胞因子的影響,同時(shí)采用Western blot和細(xì)胞免疫熒光等方法檢測(cè)NFκB磷酸化及核轉(zhuǎn)位的情況,以明確THP-1細(xì)胞產(chǎn)生炎癥反應(yīng)的胞內(nèi)信號(hào)通路；進(jìn)一步利用抑制劑阻斷TLR-4信號(hào)通路,了解其對(duì)SSL5-PMPs促進(jìn)THP-1細(xì)胞炎癥反應(yīng)的影響。研究結(jié)果1.SSL5激活人血小板并產(chǎn)生血小板(SSL5-PMPs).①重組SSL5具有生物學(xué)活性,可用于后續(xù)實(shí)驗(yàn)；②SSL5導(dǎo)致人外周血中PMPs的數(shù)量明顯增加；③ SSL5-PMPs的直徑為0.1～1 μm, PS外暴露于膜表面,表達(dá)血小板特異性的糖蛋白GPⅡb以及與細(xì)胞間相互作用的信號(hào)分子P-選擇素和CD40L。2. SSL5-PMPs促進(jìn)人單核細(xì)胞的致炎癥效應(yīng)。①SSL5不影響THP-1細(xì)胞的活力,排除因SSL5毒性作用而導(dǎo)致單核細(xì)胞產(chǎn)生炎癥反應(yīng)；② SSL5-PMPs與THP-1細(xì)胞的結(jié)合呈時(shí)間依賴性,且主要與外周血CD14++CD16+(中間型)的單核細(xì)胞結(jié)合；③ SSL5-PMPs時(shí)間和劑量依賴性的促進(jìn)單核細(xì)胞產(chǎn)生炎癥介質(zhì),包括表達(dá)和釋放IL-1β和TNFα、MCP-1以及MMP-9,并增強(qiáng)MMP-9的活性；④ ADP-PMPs和ap-PMPs也可以促進(jìn)THP-1細(xì)胞炎癥介質(zhì)mRNA的表達(dá),其水平低于SSL5-PMPs刺激的結(jié)果,而SSL5對(duì)THP-1細(xì)胞炎癥介質(zhì)mRNA的表達(dá)沒(méi)有影響；⑤ SSL5-PMPs促進(jìn)MCP-1誘導(dǎo)的單核細(xì)胞發(fā)生遷移,而SSL5則抑制遷移；3. SSL5-PMPs促進(jìn)人單核細(xì)胞炎癥反應(yīng)的機(jī)制。①阻斷CD40L與CD40的相互作用,可以部分抑制SSL5-PMPs誘導(dǎo)單核細(xì)胞產(chǎn)生炎癥介質(zhì),而抑制P-選擇素與PSGL-1的相互作用則對(duì)炎癥介質(zhì)的產(chǎn)生沒(méi)有影響；②阻斷PSGL-1導(dǎo)致SSL5-PMPs介導(dǎo)的單核細(xì)胞遷移幾乎完全被抑制,而抑制CD40L與CD40的相互作用則明顯減少單核細(xì)胞遷移的比率；③下調(diào)單核細(xì)胞CD40或TRAF6基因的表達(dá),導(dǎo)致 SSL5-PMPs誘導(dǎo)單核細(xì)胞炎癥介質(zhì)的產(chǎn)生減少,并抑制NFκB p65亞單位的磷酸化及核轉(zhuǎn)位；④阻斷TLR4信號(hào)通路對(duì)SSL5-PMPs誘導(dǎo)單核細(xì)胞釋放炎癥介質(zhì)沒(méi)有影響。研究結(jié)論SSL5,S.aureus分泌的一種毒素可以激活血小板并產(chǎn)生PMPs(SSL5-PMPs),導(dǎo)致外周血中的PMPs數(shù)量明顯增加；SSL5-PMPs與單核細(xì)胞結(jié)合,且主要與外周血中的具有促炎功能的中間型單核細(xì)胞結(jié)合,并促進(jìn)單核細(xì)胞產(chǎn)生廣泛的炎癥反應(yīng),包括增加炎性細(xì)胞因子和趨化因子的釋放、增強(qiáng)基質(zhì)金屬蛋白酶的活性以及誘導(dǎo)遷移；在機(jī)制方面,SSL5-PMPs通過(guò)其來(lái)源的CD40L與單核細(xì)胞CD40發(fā)生直接的相互作用,激活單核細(xì)胞內(nèi)CD40-TRAF6-NFκB信號(hào)通路,從而觸發(fā)單核細(xì)胞的炎癥反應(yīng)；而SSL5本身則抑制白細(xì)胞的功能。本研究闡述了S.aureus導(dǎo)致機(jī)體產(chǎn)生炎癥反應(yīng)的一種新方式,以期從這一新的角度闡明感染負(fù)荷的致動(dòng)脈粥樣硬化機(jī)制。
[Abstract]:Background Cardiovascular disease is the leading cause of death in the world. Atherosclerosis is essentially chronic inflammation of blood vessels caused by a variety of factors. Multiple infections of pathogens, i.e. "infectious burden", are considered to be a new independent risk of atherosclerosis. Infectious factors can damage the arterial wall by direct action, triggering systemic inflammation and initiating autoimmune response, leading to the occurrence and development of atherosclerosis. S, S. aureus) is one of the common clinical pathogens. Studies have shown that S. aureus infection increases the risk of acute myocardial infarction and stroke, but the mechanism is not clear. At present, it is thought that it may be related to platelet activation, increased inflammation and blood hypercoagulability. Staphylococcal superantigen-like protein 5 (SSL5) can activate platelets, and a single intravenous injection of SSL5 can also cause thrombosis in the pulmonary arteries of mice. Therefore, we speculate that SSL5 may induce inflammation by activating platelets, and explore its mechanism to provide new experimental evidence for elucidating the role of infection load in atherosclerosis. Content 1. Using flow cytometry to identify the biological activity of recombinant SSL5, in vitro detection. The number of PMPs in peripheral blood and the surface molecules of SSL5-activated PMPs (SSL5-PMPs); the morphological characteristics of SSL5-PMPs were observed by scanning electron microscopy; SSL5-PMPs and ADP-activated PMPs (ADP-PMPs) and apoptosis-induced PMPs (ap-PMPs) were prepared in vitro as stimulants and controls in subsequent experiments. 2. Human peripheral blood mononuclear cells and THP-1 were fine. Cells were used as experimental subjects to detect the effect of SSL5 on the viability of monocytes by CCK-8 method; the binding of SSL5-PMPs to monocytes was observed by scanning electron microscopy; the binding time-dependent relationship and difference between them were further detected by flow cytometry; the production of inflammatory mediators (cytokines, chemokines and matrix metalloproteinases) and migration were used as mononuclear cells. The expression of inflammatory mediators mRNA was detected by real-time quantitative PCR, the release of cytokines and chemokines by ELISA, the activity of protease by gelatin zymogram, and the migration of cells by Transwell migration assay. To investigate the difference of PMPs-induced inflammation in monocytes and the possible pathological role of SSL5. 3. Using THP-1 cells as experimental subjects, neutralizing antibodies interfered with the binding of CD40L/CD40 and P-selectin/PSGL-1 between SSL5-PMPs and THP-1 cells, and observed the effect of neutralizing antibodies on the production of cytokines and migration of THP-1 cells. To clarify the molecular basis of interaction between SSL5-PMPs and THP-1 cells, siRNA was used to silence the gene expression of CD40 or TRAF6 in THP-1 cells and observe its effect on the production of cytokines in THP-1 cells. Results 1. SSL5 activates human platelets and produces platelets (SSL5-PMPs). 1. Recombinant SSL5 has biological activity and can be used in subsequent experiments; 2. SSL5 induces PM in human peripheral blood. (3) SSL5-PMPs with a diameter of 0.1-1 micron were exposed to the membrane surface, and expressed platelet-specific glycoprotein GP II B and the signal molecules P-selectin and CD40L.2.SSL5-PMPs interacting with each other. SSL5 did not affect the activity of THP-1 cells and excluded the inflammatory effect of SSL5 on human monocytes. The toxicity of SSL5-PMPs induces inflammation of monocytes; the binding of SSL5-PMPs to THP-1 cells is time-dependent and mainly with peripheral blood CD14 +-CD16 + (intermediate type) monocytes; and the time-and dose-dependent effects of SSL5-PMPs promote the production of inflammatory mediators by monocytes, including the expression and release of IL-1beta and TNFalpha, and MCP-1 to ADP-PMPs and ap-PMPs could also promote the expression of inflammatory mediators mRNA in THP-1 cells, which was lower than that stimulated by SSL5-PMPs. SSL5 had no effect on the expression of inflammatory mediators mRNA in THP-1 cells. _SSL5-PMPs promoted MCP-1-induced migration of monocytes, while SSL5 inhibited migration. The mechanism of 5-PMPs promoting inflammation in human monocytes is: (1) Blocking the interaction between CD40L and CD40 partially inhibits the production of inflammatory mediators in monocytes induced by SSL5-PMPs, while inhibiting the interaction between P-selectin and PSGL-1 has no effect on the production of inflammatory mediators; (2) Blocking the interaction between PSGL-1 and SSL5-PMPs almost leads to the migration of monocytes mediated by PSGL-1. Inhibiting the interaction between CD40L and CD40 significantly reduced the rate of monocyte migration; 3) Downregulating the expression of CD40 or TRAF6 gene in monocytes, resulting in the decrease of inflammatory mediators in monocytes induced by SSL5-PMPs, and inhibiting the phosphorylation and nuclear translocation of NF-kappa B p65 subunit; 4) Blocking the TLR4 signaling pathway to SSL5-PMPs. Conclusion SSL5, a toxin secreted by S. aureus, can activate platelets and produce PMPs (SSL5-PMPs), resulting in a marked increase in the number of PMPs in peripheral blood; SSL5-PMPs bind to monocytes, and mainly bind to intermediate monocytes with pro-inflammatory function in peripheral blood. Promote a wide range of inflammatory responses in monocytes, including increasing the release of inflammatory cytokines and chemokines, enhancing the activity of matrix metalloproteinases and inducing migration; in mechanism, SSL5-PMPs interact directly with monocyte CD40 through CD40L, activating CD40-TRAF6-NF-kappa B signaling in monocytes. This study elucidates a new way that S. aureus causes inflammation in the body, in order to elucidate the atherosclerotic mechanism of infection load from this new perspective.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R54

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Rosa Sessa;Marisa Di Pietro;Simone Filardo;Ombretta Turriziani;;Infectious burden and atherosclerosis: A clinical issue[J];World Journal of Clinical Cases;2014年07期

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本文編號(hào)：2250416