PLC信号调控BoHV-1感染及炎症反应的分子机理研究

发布时间：2018-04-16 10:40

本文选题：牛疱疹病毒1型 + 磷脂酶C　；参考：《扬州大学》2017年硕士论文

【摘要】：牛疱疹病毒1型(Bovineherpesvirustype1,BoHV-1)是传染性牛鼻气管炎(Infectious bovinerhinotracheitis,IBR)的病原体。BoHV-1感染的临床症状主要表现为黏膜炎、繁殖障碍和上呼吸道炎症反应。该病毒感染引起的免疫抑制易导致其它细菌和病毒的继发性感染,从而引起牛呼吸道疾病综合征(bovinerespiratorydiseasecomplex,BRDC),最终导致高死亡率发生。该病毒广泛分布于世界各地给养牛业带来巨大经济损失。炎症反应在BoHV-1感染和BRDC中起重要的作用,目前BoHV-1感染诱导炎症产生的报道较少。本研究通过研究BoHV-1感染如何调控炎症相关的细胞信号MAPK通路(mitogen-activated protein kinase pathway)的活性和炎性介质分子 ROS(reactive oxidative species)的产生,来初步揭示该病毒诱导炎症反应发生的分子机制。本研究主要内容分为以下四个部分:1.BoHV-1感染调节炎症相关介质分子ROS产生的分子机制在多种病毒感染过程中ROS对于病毒复制以及病毒诱导的炎症反应都起重要作用。本研究就BoHV-1感染MDBK细胞过程中ROS、GSH(glutathione)、线粒体膜电位(mitochondrialmembranepotential,MMP)以及ATP水平分别进行检测,发现该病毒的侵入、病毒蛋白的表达和/或DNA复制均可以提高ROS水平,过量产生的ROS有助于BoHV-1复制。BoHV-1感染导致细胞内GSH水平显著降低,这可能是介导ROS产生的分子机制之一。加入外源性GSH可以显著抑制病毒复制,GSH与抗疱疹病毒药物阿昔洛韦(Acyclovir,ACV)联合使用具有协同的抗病毒作用。BoHV-1感染MDBK细胞中通过显著降低MMP和ATP水平,从而对线粒体造成损伤。有趣的是抑制ROS水平能够有效的恢复BoHV-1感染导致MMP降低的现象。本研究表明BoHV-]感染依赖ROS的产生对线粒体造成损伤,最终破坏ATP的生成是该病毒感染潜在的致病机制之一。2.BoHV-1感染调节炎症相关信号MAPK通路已知BoHV-1感染过程中能够激活MAPK/Erk1/2通路,MAPK通路除了 ERK1/2信号之外,还包括p38MAPK和JNK两个信号,它们在多种因素诱导的炎症反应中都起重要作用。BoHV-1感染是否调控p38MAPK和JNK信号,目前未见报道。本研究首次发现BoHV-1感染MDBK细胞的过程中能够激活p38MAPK和JNK信号通路以及JNK的下游信号c-Jun,另外JNK信号的激活有助于病毒复制。BoHV-1感染激活MAPK信号通路并不依赖ROS的产生,这不同于单纯疱疹病毒1型(herpes simplex virus 1,HSV-1)。本研究表明BoHV-1感染过程调控MAPK通路,该调控过程与ROS产生无关。3.PLC信号调控多因素诱导的炎症反应磷脂酶C家族(phospholipase C,PLC)/Ca2+信号调控多种生物学过程。单核/巨噬细胞在炎症反应过程起重要作用。人单核细胞系U937细胞在phorbol-12-myristate-13-acetate(PMA)刺激下可以分化成巨噬样细胞dU937。本研究以U937细胞为模型,研究PLC信号在单核细胞向巨噬细胞分化、LPS和流感病毒H1N1诱导的促炎性细胞因子表达中发挥的作用。研究发现PLC抑制剂U73122能显著抑制PMA诱导的细胞粘附相关信号Pyk2和paxillin的表达和活性,从而抑制单核细胞的粘附;U73122能显著降低巨噬细胞表面marker CD163的表达。可见PLC信号调控PMA诱导的单核细胞U937向巨噬细胞的分化。LPS和流感病毒H1N1都能诱导促炎性细胞因子的大量表达从而介导炎症反应。本研究发现U73122能显著抑制LPS刺激dU937细胞和小鼠腹腔巨噬细胞所诱导的促炎性细胞因子TNF-α和IL-1β的表达;U73122也能显著抑制流感病毒H1N1感染dU937细胞诱导的细胞因子TNF-α,IL-6,MIP-1α的表达、ROS产生,此外PLC信号还与NF-κB信号活化有关。这表明PLC信号参与多因素诱导的炎症反应。4.BoHV-1感染调控PLC信号及PLC介导的炎症反应鉴于上述研究发现PLC信号调控多因素诱导的促炎性细胞因子和炎性介质分子的表达,及炎性相关信号NF-κB的活性,而BoHV-1感染过程中是否调节PLC信号,目前并不清楚。本研究发现BoHV-1感染激活PLC有利于病毒复制,激活的PLC信号介导ROS产生及p38MAPK和Erk1/2信号的激活。表明PLC信号在BoHV-1感染过程中起重要作用,病毒促进ROS产生和激活p38MAPK和Erk1/2信号在一定程度上依赖于PLC信号。推测PLC信号对于BoHV-1感染以及病毒诱导的炎症反应可能起重要作用。
[Abstract]:Bovine herpesvirus type 1 (Bovineherpesvirustype1, BoHV-1) is an infectious bovine rhinotracheitis (Infectious bovinerhinotracheitis, IBR) the clinical symptoms of the pathogen.BoHV-1 infection mainly manifested as mucositis, reproductive disorders and upper respiratory tract inflammation. Inhibition of easily lead to secondary infection of other bacteria and viruses caused by immune the virus causing bovine respiratory the disease syndrome (bovinerespiratorydiseasecomplex, BRDC), and finally lead to high mortality. The virus is widely distributed in the world to the cattle industry to bring huge economic losses. The inflammatory reaction plays an important role in BoHV-1 infection and BRDC infection, currently reported less inflammation induced by BoHV-1 generated. This research is how to control the inflammation related cell signal MAPK passage through the infection of BoHV-1 (mitogen-activated protein kinase pathway) and the activity of inflammatory mediators ROS (reactive oxidative species) generation, to reveal the molecular mechanism of the virus induced inflammation. The main contents of this study includes the following four parts: 1.BoHV-1 infection related molecular mechanisms that regulate the inflammatory medium molecule ROS produced in a variety of virus infection in ROS has played an important role in virus replication and virus induced inflammatory reaction this study. BoHV-1 infection of MDBK cells in ROS, GSH (glutathione), mitochondrial membrane potential (mitochondrialmembranepotential, MMP) and ATP levels were detected and found the virus invasion, the expression of viral proteins and / or DNA replication can improve the level of ROS, excessive production of ROS contribute to the replication of BoHV-1.BoHV-1 infection leads to the intracellular level of GSH decreased significantly, which may be one of the molecular mechanisms mediated by ROS. Exogenous GSH can significantly inhibit virus replication, GSH and anti herpes drugs A Silowe (Acyclovir, ACV) combined with antiviral effect of.BoHV-1 co infection of MDBK cells by MMP and ATP levels decreased significantly, resulting in damage to the mitochondria. Interestingly the inhibition of ROS can effectively restore BoHV-1 infection leads to the decrease of MMP. The results indicated that BoHV-] infection on ROS the damage to the mitochondria, to generate the final damage ATP the virus infection is one of the pathogenic mechanisms underlying.2.BoHV-1 infection can activate MAPK/Erk1/2 pathway in regulation of inflammation related signaling pathway known MAPK BoHV-1 infection process, in addition to the ERK1/2 signal pathway of MAPK, including p38MAPK and JNK two signal are inflammatory reaction induced by them in a variety of factors the important role of whether.BoHV-1 infection control of p38MAPK and JNK signal, has not been reported. This is the first study found that BoHV-1 infected with M Can activate p38MAPK and JNK signaling pathway and downstream of JNK signal c-Jun DBK cells, and the activation of JNK signaling contributes to the replication of virus infection of.BoHV-1 activation of MAPK signaling pathway is not dependent on the production of ROS, which is different from the herpes simplex virus type 1 (herpes simplex virus 1, HSV-1). The results indicated that BoHV-1 infection process regulation of the MAPK pathway and the regulation process of ROS to produce inflammation independent phospholipase C.3.PLC signaling induced by multi factor family (phospholipase C, PLC) /Ca2+ signaling regulates many biological processes. Monocytes / macrophages play an important role in the inflammatory reaction process. Human monocytic U937 cells in phorbol-12-myristate-13-acetate (PMA) can stimulate in this study, differentiate into macrophage like cells dU937. to U937 cells as a model, the research of PLC signal differentiation to macrophages in mononuclear cells, LPS and H1N1 induced by influenza virus Play the proinflammatory cytokine expression. The study found that the expression and activity of PLC inhibitor U73122 can significantly inhibit PMA induced cell adhesion related signal Pyk2 and paxillin, thereby inhibiting the adhesion of monocytes; U73122 can significantly reduce the expression of marker CD163. A large number of macrophage surface expression of monocyte U937 visible PLC signaling PMA to induce macrophage differentiation of.LPS and H1N1 influenza virus can induce proinflammatory cytokine which mediates inflammatory reaction. The study found that U73122 can significantly inhibit LPS induced dU937 cells and mouse peritoneal macrophages stimulated by inflammatory cytokines TNF- alpha and IL-1 beta U73122 expression also significantly inhibited; influenza H1N1 virus infection of dU937 cells induced by cytokines TNF-, IL-6, expression of MIP-1 alpha ROS, PLC and NF- in signal kappa B signaling activation. This indicates that the PLC signal .4.BoHV-1 is involved in the inflammatory response induced by infection factors regulating inflammatory response mediated by PLC signal and PLC in view of the study found that the expression of PLC signal regulation of proinflammatory cytokines and inflammatory mediators induced by multiple molecular factors, and inflammatory related signal NF- kappa B activity, and BoHV-1 whether PLC signal regulated in dyeing process at present, is not clear. This study found that BoHV-1 infection activates PLC to virus replication, activation of PLC signaling mediated by activated ROS and p38MAPK and Erk1/2 signals. The results indicated that PLC signal plays an important role in the process of BoHV-1 infection, the virus promotes ROS production and activation of p38MAPK and Erk1/2 signal depends on the PLC signal. That PLC signal for BoHV-1 infection and virus induced inflammatory reaction may play an important role.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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