NLRP3炎症小体在汉滩病毒感染THP-1细胞中的作用

发布时间：2018-03-31 10:11

本文选题：炎症小体　切入点：汉滩病毒　出处：《第四军医大学》2014年博士论文

【摘要】：汉滩病毒（hantaan virus, HTNV）是引起我国肾综合征出血热（hemorrhagic feverwith renal syndrome, HFRS）最主要的病原体。HFRS主要表现为发热、出血、低血压休克、肾脏衰竭和多脏器功能不全综合征，自1955年首例发病报告以来，发病率和病死率居高不下，一直是严重威胁我国人民生命健康的严重公共安全问题。HFRS的发病机制尚未完全阐明，目前认为病毒对内皮细胞的直接损伤以及机体的免疫病理损害均参与了HTNV的致病过程，尤其是机体免疫系统中免疫复合物的形成、炎性因子的大量分泌（炎症风暴）、细胞毒性T细胞的杀伤作用在HFRS的发病机制中扮演了极其重要的角色。炎症小体（inflammasomes）是近年来发现的一类位于细胞内、可以引起炎症细胞因子IL-1β/IL-18分泌、继而诱发炎症反应的多聚蛋白复合体。炎症小体识别的配体种类非常广泛，涵盖了细菌、病毒、脂肪酸、结晶结构等，是机体固有免疫系统中重要的成份，也是机体细胞抵抗外源病原体感染和消除内源危险因子刺激的首要免疫屏障。目前研究已知，多种病毒感染靶细胞后均可以激活炎症小体，诱发局部的炎症反应以及后续的免疫反应。与此同时，病毒也使用了多种策略来逃逸炎症小体的识别，从而使自身能够在细胞内复制增殖。由于HTNV能够刺激内皮细胞和单核细胞分泌IL-1β，因此，HTNV很有可能也激活了细胞内的炎症小体。因此，，我们检测了HTNV感染THP-1细胞后IL-1β的分泌，以及NLRP3炎症小体相关基因和蛋白水平的变化，同时评价了Ca2+在HTNV感染过程中的作用。结果如下： 1.我们首先构建了HTNV-cRNA标准品，建立了荧光实时定量PCR检测HTNV拷贝数的方法，所得到的标准曲线参数理想，具有良好的线性关系。通过分析扩增曲线和融解曲线，发现该方法具有良好的敏感性、特异性以及重复性，用其检测THP-1细胞培养上清中HTNV的病毒载量，为后续实验奠定了基础。 2. HTNV感染THP-1细胞后，可以引起pro-IL-1β蛋白表达的增加以及IL-1β的分泌，并且pro-IL-1β蛋白表达的水平和IL-1β的分泌量与感染的病毒量呈正比。同时，IL-1β的分泌需要病毒复制和增殖过程的诱导，单纯的病毒核酸及蛋白刺激均不能引起IL-1β分泌。 3. HTNV感染THP-1细胞后，可以引起炎症相关基因的表达增高，特别是NLRP3炎症小体基因的表达增高，并且NLRP3炎症小体相关蛋白的表达也增高，我们还用激光共聚焦显微镜检测了NLRP3炎症小体在HTNV感染THP-1细胞后的定位情况，分别从基因和蛋白水平证实了HTNV感染可以引起NLRP3炎症小体的激活。此外，我们使用NLRP3siRNA和caspase-1抑制剂，进一步验证了NLRP3炎症小体在HTNV感染中的作用。 4.我们还检测了活细胞内游离Ca2+在THP-1细胞中的变化情况，发现HTNV感染THP-1细胞会引起细胞内Ca2+浓度增高，螯合细胞外Ca2+会导致IL-1β分泌的减少，提示Ca2+有可能参与了NLRP3炎症小体的激活过程。综上所述，我们发现HTNV感染THP-1细胞可以激活NLRP3炎症小体，增加细胞内游离的Ca2+的浓度，启动下游caspase-1和IL-1β信号通路，最终导致IL-1β的分泌，诱发局部的炎症反应。本研究为探索HTNV感染靶细胞的具体过程和机制提供了一些新的证据和思路。
[Abstract]:Hantaan virus ( HTNV ) is one of the most important pathogens causing hemorrhagic fever with renal syndrome in China .

Inflammatory small body ( IL - 1尾 / IL - 18 ) is an important component in the innate immune system , and it is an important component in the innate immune system . It is also a primary immune barrier against exogenous pathogen infection and the elimination of endogenous risk factors .

Because HTNV can stimulate the secretion of IL - 1尾 in endothelial cells and monocytes , HTNV may also activate the inflammatory cells in cells . Therefore , we have detected the secretion of IL - 1尾 in HTNV infected THP - 1 cells , as well as changes in gene and protein levels associated with NLRP3 inflammation , and also evaluated the role of Ca2 + in HTNV infection . The results are as follows :

1 . We first constructed the HTNV - cRNA standard , established the method of detecting HTNV copy number by fluorescence real - time quantitative PCR . The obtained standard curve parameter is ideal and has a good linear relationship . By analyzing the amplification curve and the melting curve , it is found that the method has good sensitivity , specificity and repeatability , and it can be used to detect the viral load of HTNV in THP - 1 cell culture supernatant .

2 . When HTNV infected THP - 1 cells , the expression of pro - IL - 1尾protein and secretion of IL - 1尾can be induced , and the level of pro - IL - 1尾protein expression and the secretion of IL - 1尾are directly proportional to the amount of viral infection . At the same time , IL - 1尾secretion requires the induction of viral replication and proliferation , and only viral nucleic acid and protein stimulation can not induce IL - 1尾 secretion .

3 . After the infection of THP - 1 cells with HTNV , the expression of inflammation - related genes increased , especially the expression of NLRP3 - inflammatory small - body gene was increased , and the expression of NLRP3 - associated protein was increased .

4 . We also detected the changes of free Ca2 + in THP - 1 cells , and found that HTNV infected THP - 1 cells could increase intracellular Ca 2 + concentration , and Ca2 + could decrease the secretion of IL - 1尾 , suggesting that Ca2 + might participate in the activation of NLRP3 inflammatory cells .

In conclusion , we find that HTNV infected THP - 1 cells can activate NLRP3 inflammatory cells , increase intracellular free Ca2 + concentration , activate downstream caspase - 1 and IL - 1尾 signaling pathways , eventually lead to the secretion of IL - 1尾and induce local inflammatory responses . This study provides some new evidence and ideas for exploring the specific processes and mechanisms of HTNV infected target cells .

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.8

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