發(fā)布時(shí)間：2018-08-22 20:49

【摘要】：肝臟缺血再灌注(Ischemia/Reperfusion, I/R)損傷是肝臟外科手術(shù)中不可避免的病理生理過(guò)程。缺血器官早期大量中性粒細(xì)胞浸潤(rùn),蛋白水解酶釋放,氧自由基大量產(chǎn)生以及細(xì)胞因子激活等導(dǎo)致組織損傷,啟動(dòng)肝細(xì)胞的死亡與固有免疫應(yīng)答,是發(fā)生急性再灌注損傷的重要原因。隨著對(duì)肝內(nèi)免疫微環(huán)境的重新認(rèn)識(shí),研究者發(fā)現(xiàn)這些激發(fā)損傷作用的因素卻是啟動(dòng)后期肝臟修復(fù)的重要因素。如何界定其作用和尋求合適的干預(yù)時(shí)機(jī)是目前肝臟I/R損傷以及后期修復(fù)研究的熱點(diǎn)和難點(diǎn)。IL-17A已被證實(shí)是肝臟I/R損傷過(guò)程中重要炎的癥因子,而新近的研究發(fā)現(xiàn)IL-17A同時(shí)也是參與組織修復(fù)(肝臟、神經(jīng)、小腸粘膜和骨等)的重要啟動(dòng)因子。在肝臟I/R損傷早期,IL-17A可由以下細(xì)胞分泌：CD4+T細(xì)胞、NKT細(xì)胞、γδT細(xì)胞、NK細(xì)胞和中性粒細(xì)胞。目前,對(duì)肝臟I/R后確切的IL-17A+細(xì)胞亞群仍有爭(zhēng)議；但是對(duì)于減輕早期肝臟IR損傷及后期修復(fù)而言,明確具體的細(xì)胞來(lái)源具有指導(dǎo)臨床治療的價(jià)值。在肝臟I/R損傷后早期階段便促進(jìn)庫(kù)否細(xì)胞(Kupffer Cells, KCs)分泌細(xì)胞因子白介素-6、腫瘤壞死因子、肝細(xì)胞生長(zhǎng)因子等細(xì)胞因子。這些蛋白恰恰是促進(jìn)肝臟修復(fù)的經(jīng)典細(xì)胞因子,已經(jīng)被證實(shí)能夠加快肝細(xì)胞再生進(jìn)程。研究證明,IL-17A可以促進(jìn)炎癥反應(yīng),那么,IL-17A的出現(xiàn)是否能夠活化KCs,從而激活肝臟再生信號(hào)。詳細(xì)了解IL-17A信號(hào)在肝臟損傷和修復(fù)中的作用機(jī)制對(duì)于指導(dǎo)臨床治療具有重要的意義。Olrl基因編碼了凝集素樣氧化型低密度脂蛋白受體-1(Lectin-like Oxidized Low-density Lipoprotein Receptor-1, LOX-1),LOX-1是一種清道夫受體。本課題旨在圍繞IL-17A與LOX-1的相互聯(lián)系,進(jìn)一步揭示LOX-1在I/R損傷中的作用及上下游作用機(jī)制。本課題采用小鼠70%肝臟I/R(缺血90min)模型,利用IL-17A-/-和IL-17RA-/-小鼠,分析I/R后缺乏IL-17A信號(hào)時(shí)肝臟損傷和再生的變化。博士研究生期間主要獲得以下結(jié)果：1.小鼠肝臟I/R損傷后急性期,即I/R后24小時(shí)內(nèi),IL-17A/IL-17RA信號(hào)的缺失會(huì)減輕I/R造成的肝臟損害和炎癥反應(yīng)；后期,約在損傷后48小時(shí)-72小時(shí),IL-17A/IL-17RA信號(hào)的缺失削弱了肝臟的修復(fù)進(jìn)程。2.I/R損傷早期,IL-17A加重肝臟炎癥反應(yīng)可能與肝臟內(nèi)大量中性粒細(xì)胞的浸潤(rùn)相關(guān)。而此階段,中性粒細(xì)胞成為肝內(nèi)IL-17A的主要來(lái)源。在損傷修復(fù)階段,肝內(nèi)IL-17A主要來(lái)源于ROR γt+細(xì)胞。這一結(jié)果為干預(yù)炎癥損傷、促進(jìn)修復(fù)提供了重要的干預(yù)時(shí)間點(diǎn)與靶點(diǎn)。3.證實(shí)LOX-1與IL-17A介導(dǎo)的肝臟I/R損傷后修復(fù)相關(guān)。其機(jī)制可能是IL-17A/IL-17RA信號(hào)通過(guò)TRAF6、NF-κB調(diào)節(jié)LOX-1的表達(dá)。在下游,LOX-1與IL-17A一起激活庫(kù)否細(xì)胞內(nèi)的絲裂原活化蛋白激酶(Mitogen-activated Protein Kinase,MAPK)信號(hào)通路。4.IL-17A信號(hào)通過(guò)LOX-1間接地活化KCs,使其釋放一系列促進(jìn)肝臟修復(fù)的正調(diào)節(jié)信號(hào),如HGF,IL-6等。綜上所述,我們的研究表明,IL-17A/IL-17RA信號(hào)通路在肝臟I/R損傷以及修復(fù)過(guò)程中發(fā)揮核心作用,一方面,中性粒細(xì)胞分泌的IL-17A可以放大急性期肝臟的炎癥反應(yīng),證明了IL-17A/IL-17RA信號(hào)在肝臟缺血再灌注損傷中居于炎癥調(diào)控網(wǎng)絡(luò)的中心地位；另一方面,循環(huán)/組織中高表達(dá)的IL-17A通過(guò)與其受體IL-17RA的結(jié)合,促進(jìn)LOX-1基因的表達(dá),直接或間接地激活下游KCs的MAPK通路相關(guān)分子的磷酸化,促進(jìn)靜止的KCs活化,并分泌HGF、.IL-6等細(xì)胞因子,促進(jìn)肝細(xì)胞的增殖,抑制其凋亡。最終促進(jìn)肝臟I/R損傷后的修復(fù)。上述這些結(jié)果有利于我們更深入地了解IL-17A在肝臟I/R損傷急性期促炎癥以及后期促修復(fù)過(guò)程中作用機(jī)制,同時(shí)也為肝臟I/R損傷的干預(yù)提供時(shí)間節(jié)點(diǎn)以及新的靶標(biāo)。
[Abstract]:Ischemia/Reperfusion (I/R) injury is an unavoidable pathophysiological process in hepatic surgery. Invasion of large numbers of neutrophils, release of proteolytic enzymes, production of oxygen free radicals and activation of cytokines in the early stage of ischemic organs lead to tissue damage, initiating death of hepatocytes and innate immune response. With the re-understanding of the intrahepatic immune microenvironment, researchers have found that these factors that trigger the injury are important factors in initiating late liver repair. How to define the role of these factors and find the right time to intervene are hot and difficult issues in the study of liver I/R injury and late liver repair. Interleukin-17A (IL-17A) has been proved to be an important inflammatory factor in liver I/R injury. Recent studies have found that IL-17A is also an important promoter of tissue repair (liver, nerve, small intestinal mucosa and bone, etc.). In the early stage of liver I/R injury, IL-17A can be secreted by CD4 + T cells, NKT cells, gamma delta T cells, NK cells and neutral cells. Granulocytes. At present, the exact subpopulation of IL-17A+ cells after liver I/R is still controversial; however, it is valuable to identify specific cell sources for clinical treatment in alleviating early liver IR injury and late repair. Kupffer Cells (KCs) secrete cytokine interleukin-6 at the early stage after liver I/R injury. Tumor necrosis factor, hepatocyte growth factor and other cytokines. These proteins are classical cytokines that promote liver repair and have been shown to speed up the process of hepatocyte regeneration. The mechanism of L-17A signaling in liver injury and repair is of great significance for guiding clinical treatment. Olrl gene encodes lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), which is a scavenger receptor. The purpose of this study is to explore the role of LOX-1 in I/R injury and its mechanism of upstream and downstream. The present study used 70% I/R (ischemia 90 min) model of mice liver and IL-17A-/- and IL-17RA-/- mice to analyze the changes of liver injury and regeneration in the absence of IL-17A signal after I/R. Deletion of IL-17A/IL-17RA signal attenuates liver damage and inflammation induced by I/R in the acute phase after injury, i.e. within 24 hours after I/R. Deletion of IL-17A/IL-17RA signal attenuates the repair process of liver in the late phase, about 48-72 hours after injury. 2. In the early phase of I/R injury, IL-17A may aggravate liver inflammation and a large number of neutral liver reactions. At this stage, neutrophils become the main source of intrahepatic IL-17A. At the stage of injury repair, intrahepatic IL-17A mainly comes from ROR gamma T + cells. This result provides an important intervening time point and target for intervening inflammatory injury and promoting repair. 3. I t is confirmed that LOX-1 and IL-17A mediated liver I/R repair phase after injury. Downstream, LOX-1, along with IL-17A, activates the mitogen-activated protein kinase (MAPK) signaling pathway in the Kupffer cells. 4. IL-17A signaling indirectly activates KCs through LOX-1 to release a series of enhancements to liver repair. In summary, our studies have shown that IL-17A/IL-17RA signaling pathway plays a central role in liver I/R injury and repair. On the one hand, IL-17A secreted by neutrophils can amplify inflammation in the acute phase of liver injury, which proves that IL-17A/IL-17RA signaling is involved in liver ischemia-reperfusion injury. On the other hand, IL-17A, which is highly expressed in circulatory/tissue, promotes the expression of LOX-1 gene by binding to its receptor IL-17RA, directly or indirectly activates the phosphorylation of MAPK pathway-related molecules in downstream KCs, promotes the activation of resting KCs, and secretes cytokines such as HGF, IL-6, and promotes hepatocyte proliferation. These results will help us to understand the role of IL-17A in promoting inflammation in the acute phase of liver I/R injury and in the late phase of liver repair, and provide time nodes and new targets for the intervention of liver I/R injury.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R657.3;R-332

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