S1PR1调控不同毒力新城疫病毒诱导炎症反应的机制研究

发布时间：2018-06-03 08:23

本文选题：NDV + 炎症反应　；参考：《华南农业大学》2016年博士论文

【摘要】：新城疫(Newcastle Disease,ND)是由新城疫病毒(Newcastle Disease Virus,NDV)引起的一种以禽类呼吸道、消化道黏膜出血为特征的急性、烈性、败血性传染病。NDV感染引起机体各组织不同程度的渗出性炎症,反映了NDV致病能力的高低,然而目前对其炎症反应的机制尚不清楚。1-磷酸鞘氨醇受体1(Sphingosine-1-phosphate receptor 1,S1PR1)是病毒诱导的炎症致病中关键的免疫调节因子,关于禽类S1PR1分子的功能研究鲜见报道,鸡S1PR1在ND炎症反应的功能值得探讨。本研究分别以强、弱NDV毒株感染鸡胚成纤维细胞(DF-1)、鸡髓样树突状细胞(m DCs)和SPF鸡为模型,通过体内和体外试验综合比较不同毒力NDV感染引起的炎症反应及S1PR1的表达差异;在此基础上,研究S1PR1调控NDV引起宿主炎症反应的分子机制,为临床上控制NDV感染引起的炎症反应及应用免疫调节性药物防治ND提供理论指导。本研究主要内容如下:1.不同毒力新城疫病毒感染引起的鸡炎症反应及鸡S1PR1基因的表达本研究首先选取2株生物学特性差异较大的NDV毒株GM株和La Sota株感染SPF鸡,观察不同毒株引起的炎症表现,并通过荧光定量PCR对促炎性细胞因子IL-1β的转录进行分析。强毒株GM株感染后表现出渗出性炎症症状,感染鸡呼吸道出现大量黏液,消化道有明显的出血点;而La Sota株仅表现出轻微的呼吸道症状。组织切片观察可见,在GM株感染鸡的脑、肺脏、脾脏和腺胃中出现出血和大量炎性细胞浸润等炎性显微病理变化,La Sota株仅有少量炎性细胞聚集。GM株在小肠、脑、法氏囊、腺胃和盲肠扁桃体中增殖显著上调,依次为27.5倍、14倍、5.6倍、3.8倍和3.5倍;La Sota株仅在脑中增殖水平较高(6.8倍)。在感染组脏器中检测到IL-1β的上调表达,GM组高达8.7倍,La Sota组达到6.4倍,GM组的表达水平达显著高于La Sota组。NDV感染后S1PR1在脑、肝脏、腺胃和法氏囊中表达量较高,最高可达6倍(脑),最低为2倍(法氏囊);在肾脏、小肠和胰腺等组织中表达下调。为研究鸡S1PR1的功能,本研究从DF-1细胞中扩增了鸡S1PR1基因,并构建了真核表达载体p CI-S1PR1,在DF-1细胞中鸡S1PR1基因得到了有效的表达。2.S1PR1参与调控NDV诱导的炎症反应为探究S1PR1与NDV炎症的关系,本研究检测了NDV感染后DF-1细胞中炎性细胞因子及S1PR1的表达:GM感染后IL-1β、IL-6和IL-18表达显著上调,依次达22倍、41倍和5.5倍,La Sota株感染组与对照组无显著差异,仅有2倍、1.2倍和1.5倍上调;GM感染诱导IL-8和CCL5 48倍和9.2倍的上调表达,La Sota株感染组也有8.5倍和4.3倍上调表达,显著高于对照组;TNF-α在La Sota组后呈2.8倍的上调表达,而GM组表达下调。GM株感染引起S1PR1基因2.5倍的上调表达,而La Sota株感染后S1PR1的表达水平无显著差异;NDV感染3 h起,可在感染细胞中检测到S1PR1的蛋白表达,且GM组表达水平高于La Sota组。使用W146抑制S1PR1基因表达后,显著下调IL-6(40倍)和IL-1β(10倍)的转录和160 pg/m L、250 pg/m L蛋白表达。S1PR1过表达显著上调NDV诱导的IL-1β的基因转录(5倍)和蛋白表达水平(250 pg/m L),下调IL-6的表达210 pg/m L。抑制或过表达S1PR1基因不影响NDV的复制与增殖。3.S1PR1调控新城疫病毒感染引起炎性反应的信号通路研究为进一步研究S1PR1对NDV炎症反应的调控机制,选择与诱导IL-1β表达相关的核转录因子NF-κB开展研究。本研究利用NDV强毒GM株感染DF-1细胞,在GM株感染早期Rel A/p65的表达水平显著上调,在感染3 h其m RNA水平达到6.7倍,至感染6 h达到最高峰(10.8倍),后呈下降趋势,感染24 h其表达水平与未感染组无显著差异;NDV感染后细胞荧光素酶值显著上调,高于对照组12倍;在感染3 h其蛋白表达水平最高,随着感染时间的延长逐渐降低。W146处理后,NDV感染引起的NF-κB转录显著下调,比DMSO处理组降低了10倍;其荧光素酶值显著下调了1.5倍。此外,W146处理组NF-κB的蛋白表达水平和亚细胞定位情况与DMSO处理组无显著差异。由于NF-κB的激活还需要IκB蛋白的磷酸化等过程,因此,本研究对于S1PR1能否调控NF-κB信号通路还不能提供明确的结论,具体通路有待研究。4.不同毒力NDV诱导的鸡DC细胞炎症反应为了更加全面地研究NDV感染引起的鸡炎症反应,本研究选择具有抗原递呈功能的树突状细胞(DC)作为模型,研究NDV在其中的炎性反应。首先从雏鸡骨髓中分离单核细胞,利用GM-CSF和IL-4在体外诱导分化成DC。根据细胞分化周期和病毒感染特性,在细胞培养第3天分别接种NDV毒株GM株和La Sota株,检测NDV复制以及炎性细胞因和S1PR1的表达。结果显示,NDV在DC中能够有效复制并形成细胞病变,GM株最高滴度可达107.2 TCID50/100μL,与La Sota株最高峰值差约为10 000倍。GM组感染后IL-1β(10.2倍)、IFN-β(7.6倍)和CCL5(35.4倍)的表达显著上调;而La Sota株诱导IL-10表达(峰值为7.5倍),上调CCL5表达(14.3倍)。S1PR1基因在DC细胞中呈下调表达。本研究从NDV感染过程中机体免疫和病毒感染的动态变化出发,结合对免疫调节分子S1PR1的研究,对NDV感染导致机体渗出性炎症的分子机制进行研究。主要发现了S1PR1分子在NDV感染禽类的炎症反应中具有调节细胞因子表达水平的作用,丰富了NDV致病机制的内容,为临床上控制NDV感染引起渗出性炎症及应用免疫调节性药物防治ND提供科学的理论指导。
[Abstract]:Newcastle Disease (ND) is a kind of acute, acute, severe, severe, septicemia,.NDV infection, caused by Newcastle disease virus (Newcastle Disease Virus, NDV), which is characterized by avian respiratory tract and digestive tract mucous mucous mucous membrane, which causes the tissues of different degrees of exudative inflammation in the body, which reflects the degree of NDV pathogenicity. However, it is currently inflamed. The mechanism of the disease reaction is not yet clear that.1- sphingosine receptor 1 (Sphingosine-1-phosphate receptor 1, S1PR1) is the key immunomodulatory factor in the virus induced inflammatory disease. The function of avian S1PR1 molecule is rarely reported. The function of chicken S1PR1 in ND inflammation is worthy of discussion. This study infect chickens with strong and weak NDV strains respectively. Embryo fibroblasts (DF-1), chicken medullary dendritic cells (m DCs) and SPF chickens were used as models. Through in vivo and in vitro experiments, the inflammatory response and the difference of S1PR1 expression caused by different virulence NDV infection were compared. On this basis, the molecular mechanism of S1PR1 regulating NDV caused by host inflammation was studied to control the inflammation caused by NDV infection in clinical. The main contents of this study were as follows: 1. the main contents of this study were as follows: 1. the inflammatory response of chickens caused by different virulent Newcastle disease virus infection and the expression of chicken S1PR1 gene were first selected to select 2 NDV strains of NDV strain and La Sota strain infected with SPF chickens, and to observe the causes of different strains. Inflammation, and analysis of the transcription of proinflammatory cytokine IL-1 beta through fluorescence quantitative PCR. The GM strain of the virulent strain showed an exudative inflammatory symptom, a large number of mucus appeared in the respiratory tract of the chicken, and the digestive tract had obvious bleeding points, while the La Sota strain showed only slight respiratory symptoms. Tissue section observation showed that the sense of GM strain was found. There were inflammatory micropathological changes in the brain, lungs, spleen and glandular stomach of the chicken, and only a small number of inflammatory cells in La Sota strain increased significantly in the small intestine, the brain, the bursa of the Fabricius, the glandular stomach and the cecal tonsil, which were 27.5 times, 14 times, 5.6 times, 3.8 times and 3.5 times of the cecal tonsil, and the La Sota strain only proliferated in the brain. Higher level (6.8 times). The expression of IL-1 beta was detected in the infection group, the GM group was 8.7 times higher and the La Sota group reached 6.4 times. The expression level of GM group was significantly higher than that of La Sota group.NDV infection. The expression of S1PR1 in the brain, liver, gland stomach and bursa was higher, up to 6 times (brain), and the lowest was 2 times (bursa of Fabricius); in the kidney, the small intestine and pancreas In order to study the function of chicken S1PR1, the chicken S1PR1 gene was amplified from DF-1 cells and the eukaryotic expression vector p CI-S1PR1 was constructed. The S1PR1 gene of chicken in DF-1 cells was effectively expressed in the S1PR1 gene to participate in the regulation of NDV induced inflammatory reaction to explore the relationship between S1PR1 and NDV inflammation. This study detected NDV The expression of inflammatory cytokines and S1PR1 in DF-1 cells after infection: the expression of IL-1 beta, IL-6 and IL-18 increased significantly after GM infection, which were 22 times, 41 times and 5.5 times in turn. There was no significant difference between the La Sota strain group and the control group, only 2 times, 1.2 times and 1.5 times up; GM infection induced IL-8 and CCL5 48 times and 9.2 times up to up expression, and La isolates also 8 The up-regulated expression of.5 and 4.3 times was significantly higher than that in the control group; TNF- alpha was up to up expression 2.8 times after La Sota, and the expression of S1PR1 gene was up regulated by down regulated.GM strain in GM group, but there was no significant difference in the expression level of S1PR1 in La Sota strain. NDV infection 3 h, and can detect the protein expression in infected cells. The expression level of the group was higher than that of the La Sota group. After the expression of S1PR1 gene was inhibited by W146, the transcription of IL-6 (40 times) and IL-1 beta (10 times) and 160 pg/m L were significantly lowered. The expression of 250 pg/m L protein expressed significantly up regulation of the gene transcriptional (5 times) and protein expression (250). The S1PR1 gene does not affect the signaling pathway of the replication and proliferation of NDV by.3.S1PR1 regulation of the inflammatory response induced by NDV infection. The study of the regulation mechanism of S1PR1 on NDV inflammation is to select the nuclear transcription factor NF- kappa B related to the induction of IL-1 beta expression. This study uses NDV virulent GM strains to infect DF-1 cells and in GM strains The expression level of Rel A/p65 in early infection was significantly up, 6.7 times the level of M RNA in the infection 3 h, to the peak of infection 6 h (10.8 times), and then decreased, and the expression level of infection 24 h was not significantly different from that in the uninfected group. After NDV infection, the value of luciferase was up significantly up to 12 times higher than that of the control group; and 3 h protein expressed in the infection of 3 h. With the gradual decrease of.W146 treatment, the NF- kappa B transcription caused by NDV infection was down significantly down, 10 times lower than that of DMSO treatment group, and the value of its luciferase was significantly reduced by 1.5 times. In addition, there was no significant difference between the protein expression level of NF- kappa B and the subcellular localization of the W146 treatment group with the DMSO treatment group. Due to the NF- kappa B excitation It also requires the process of phosphorylation of I kappa B protein. Therefore, this study does not provide a clear conclusion on whether S1PR1 can regulate the NF- kappa B signaling pathway. The specific pathway needs to be studied in the study of the inflammatory response of chicken DC cells induced by.4. with different virulence NDV in order to study the inflammatory response of chicken caused by NDV infection more comprehensively. A functional dendritic cell (DC) was used as a model to study the inflammatory response of NDV. First, mononuclear cells were isolated from chicken bone marrow, and GM-CSF and IL-4 were used to differentiate into DC. according to the cell differentiation cycle and virus infection characteristics. NDV strain GM strain and La Sota strain were planted for third days in cell culture, and NDV replication and inflammation were detected. The results showed that NDV could effectively copy and form cell lesions in DC, and the highest titer of GM strain could reach 107.2 TCID50/100 mu L, and the highest peak value of La Sota strain was about 10000 times.GM group, and IL-1 beta (10.2 times), IFN- beta (7.6 times) and CCL5 (35.4 times). The value is 7.5 times), up regulation of CCL5 expression (14.3 times).S1PR1 gene is down expression in DC cells. This study, based on the dynamic changes in the immune and viral infection of NDV, combined with the study of the immunomodulatory molecule S1PR1, studied the molecular mechanism of the exudative inflammation of the body caused by NDV infection. The main discovery of S1PR1 molecule in N The inflammatory response of DV infected birds has a role in regulating the expression of cytokines, enriching the content of the pathogenesis of NDV, providing scientific theoretical guidance for the clinical control of exudative inflammation caused by NDV infection and the application of immunoregulatory drugs to prevent and control ND.
【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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