IL-17A在肝脏缺血再灌注损伤及肝硬化发生中的作用机制研究

发布时间：2018-06-27 18:53

本文选题：IL-17A + 肝缺血再灌注损伤　；参考：《南京医科大学》2014年博士论文

【摘要】：肝脏疾病是危害我国人民健康的重大疾病之一,其长病程、难治性以及相对不良的预后,一直对公共医疗资源的分配和患者的生存质量造成极大的影响。肝脏缺血再灌注损伤是肝脏手术、肝移植或休克等的常见并发症,其诱发的免疫反应以及释放的免疫因子会导致患者肝脏以及肺、肠等远隔脏器功能的损伤,影响术后各器官功能的恢复,最终增加患者的围手术期死亡风险。同时,慢性肝炎病毒感染导致的非可控性肝炎被认为是肝硬化发生的最主要原因之一。持续性肝细胞死亡、炎症细胞浸润、肝脏代偿性增生以及肝小叶重塑则被普遍认为是肝硬化发展的病理过程。因此,从炎症因子以及相关分子信号通路的层面认识肝脏缺血再灌注损伤发生机制以及慢性炎症-肝硬化转化的过程有助于阐述上述疾病发生的机理,并提供潜在的临床干预手段。 IL-17A作为一种新近发现的细胞因子,首先被证实来源于Th17细胞,同时IL-1β, IL-6, TGF-β可以作为独立的因素,参与Th17细胞的成熟,而IL-23则被认为是促进Th17细胞分化、维持Th17功能的重要细胞因子。之后的一些基于动物模型和体外实验的研究也表明,IL-17A可能由NKT、Paneth细胞等细胞分泌。IL-17A信号通路在调控炎症极早期炎性细胞(中性粒细胞、淋巴细胞等)向组织间浸润、诱导新生血管生成甚至自身免疫系统疾病具有重要作用,且IL-17A信号通路的激活与肝脏肿瘤的发生也有重要联系。但目前为止,IL-17A在临床肝脏缺血再灌注损伤中的作用还并未得到临床标本的验证,且IL-17A在肝硬化中的作用机制也未见诸报道,因此,本研究分别以临床标本为基础,结合动物模型及体外实验,系统地研究了IL-17A及其相关信号通路在肝脏缺血再灌注损伤以及肝脏慢性炎症促进肝硬化发生发展中的作用及其机制。博士研究生期间主要获得以下结果： 1.肝脏缺血再灌注损伤初期,肝细胞受损释放的大量DAMPs诱导Kupffer's细胞中的NF-κB活化,随后,IL-1β、IL-6、 IL-23等细胞因子的大量释放参与并调节机体免疫反应,并有可能促进Th17细胞的分化。因此,探讨肝脏缺血再灌注损伤中IL-17A的作用及其与IL-1等细胞因子间的crosstalk显得十分重要。利用部分肝切除术患者术中需阻断肝门这一特点,我们收集了肝门阻断后再开放不同时间的患者外周血,结果发现IL-17A在外周血中表达明显增高。利用法国国家科学研究中心分子免疫与胚胎研究所Dr. Bernhard Ryffel教授提供的IL-1R1基因敲除小鼠,我们建立70%缺血再灌注模型,发现肝脏组织中的IL-17A依赖于IL-1R1通路的激活,同时,针对IL-17RA基因敲除小鼠的相关实验也证明IL-17A不仅介导了肝损伤更直接诱导了肝脏炎症细胞浸润。更为重要的是,我们发现IL-17A主要来源于中性粒细胞并依赖细胞内RORγt的激活,而中性粒细胞释放IL-17A可能与IL-23的刺激有关。因此,我们在国际上首先提出,RORγt依赖的中性粒细胞可以通过释放IL-17A诱导更多中性粒细胞进入炎症区域,进而放大缺血再灌注损伤。我们也发现,通过阻断IL-1R1、IL-17A或中性粒细胞,可以有效减轻小鼠肝功能损伤和炎症水平,为临床干预缺血再灌注损伤,保护患者肝功能提供了潜在的靶点。 2.众所周知,多种因素导致的慢性炎性损伤最终会导致肝纤维化。前述研究中已经证明IL-17A可以促进中性粒细胞向炎症组织中募集,在肝缺血再灌注损伤中起着放大炎症的作用,而IL-17A在慢性炎症向肝脏纤维化转化这一过程中的作用还未见报道。因此,通过反复腹腔注射CC14,我们成功建立了小鼠肝硬化模型,发现在慢性肝脏损伤及肝硬化的发生过程中,IL-17A信号通路的敲除可以明显的减少肝脏炎症细胞浸润,肝组织损伤以及肝纤维组织沉积。在临床标本中,我们也发现IL-17A与肝纤维化显著相关。通过分离小鼠原代肝星状细胞并进行一系列体外实验,我们证明在肝硬化的发展过程中,IL-17A可以直接作用于星状细胞并导致其激活、释放大量胶原纤维。进一步针对其机制的相关研究也表明,IL-17A通过MAPK通路,促使p38、 ERK磷酸化,促进静止的肝星状细胞向分泌型成纤维细胞转化,从而参与了肝硬化的发生和发展。综上所述,我们研究表明,IL-17A/IL-17RA信号通路在肝脏急慢性炎症损伤以及肝硬化发生过程中发挥核心作用,一方面,中性粒细胞分泌的IL-17A可以放大局部的炎症反应,证明了IL-17A在肝脏缺血再灌注损伤以及CC14诱导肝脏损伤中居于炎症调控网络的中心地位；另一方面,循环／组织中高表达的IL-17A通过与其受体IL-17RA的结合,激活下游MAPK通路相关分子的磷酸化,促进静止的星状细胞向活化的成纤维细胞转化,并分泌TGF-β、IL-6等细胞因子和细胞外基质、胶原纤维,促进肝脏发生纤维化、肝小叶重塑,最终导致肝硬化的病理学改变。上述这些结果有利于我们更深入了解IL-17A在肝脏炎症以及肝纤维化过程中的作用机制,同时也为上述疾病的早期治疗和病程监控提供了新靶标。
[Abstract]:Liver disease is one of the major diseases which endanger the health of our people . It has a long history , difficult treatment and relatively poor prognosis . The liver ischemia - reperfusion injury is a common complication of liver surgery , liver transplantation or shock .

IL - 17A , as a newly discovered cytokine , is first confirmed to be derived from Th17 cells , while IL - 1尾 , IL - 6 , TGF - 尾 can be used as an independent factor to participate in the maturation of Th17 cells .

The following results are mainly obtained during the doctoral student ' s graduate student :

1 . In the early stage of liver ischemia / reperfusion injury , the activation of NF - 魏B was induced by a large amount of DAMPs released from the liver cells , and subsequently , IL - 1尾 , IL - 6 , IL - 23 and other cytokines were released to participate in and regulate the body immune response , and it was possible to promote the differentiation of Th17 cells . We have found that IL - 17A is mainly derived from neutrophils and is dependent on the activation of IL - 1R1 pathway . At the same time , we find that IL - 17A is mainly derived from neutrophils and is dependent on the activation of IL - 23 . Therefore , we have found that IL - 17A can induce more neutrophils to enter the inflammatory region by releasing IL - 17A and then amplify the ischemia - reperfusion injury .

2 . It is well known that chronic inflammatory injury caused by multiple factors can eventually lead to liver fibrosis . In the above study , IL - 17A can be used to promote the recruitment of neutrophils to inflammatory tissues and play an important role in the pathogenesis of liver fibrosis . In the course of the development of liver cirrhosis , IL - 17A can significantly reduce inflammatory cell infiltration , liver tissue damage and liver fibrosis . In clinical specimens , we have shown that IL - 17A can directly act on stellate cells and lead to activation and release of large amounts of collagen fibers .

In conclusion , we have shown that IL - 17A / IL - 17RA signaling pathway plays a central role in liver acute and chronic inflammatory injury and cirrhosis . On the one hand , IL - 17A secreted by neutrophils can amplify the local inflammatory response , and demonstrate that IL - 17A plays a central role in hepatic ischemia - reperfusion injury and CC14 - induced liver injury .
On the other hand , the high expression of IL - 17A in circulating / tissue can activate the phosphorylation of related molecules of downstream MAPK pathway by binding to its receptor IL - 17RA , promote the transformation of quiescent stellate cells to activated fibroblasts , and secrete TGF - 尾 , IL - 6 and other cytokines and extracellular matrix , collagen fibers , promote liver fibrosis , hepatic lobule remodeling , and eventually lead to pathological changes in liver cirrhosis . These results are helpful for us to understand the mechanism of IL - 17A in the process of liver inflammation and liver fibrosis , and provide new targets for early treatment and disease course monitoring of the above diseases .
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.2

【共引文献】