發(fā)布時間：2018-06-03 08:23

  本文選題：NDV + 炎癥反應　； 參考：《華南農業(yè)大學》2016年博士論文

【摘要】：新城疫(Newcastle Disease,ND)是由新城疫病毒(Newcastle Disease Virus,NDV)引起的一種以禽類呼吸道、消化道黏膜出血為特征的急性、烈性、敗血性傳染病。NDV感染引起機體各組織不同程度的滲出性炎癥,反映了NDV致病能力的高低,然而目前對其炎癥反應的機制尚不清楚。1-磷酸鞘氨醇受體1(Sphingosine-1-phosphate receptor 1,S1PR1)是病毒誘導的炎癥致病中關鍵的免疫調節(jié)因子,關于禽類S1PR1分子的功能研究鮮見報道,雞S1PR1在ND炎癥反應的功能值得探討。本研究分別以強、弱NDV毒株感染雞胚成纖維細胞(DF-1)、雞髓樣樹突狀細胞(m DCs)和SPF雞為模型,通過體內和體外試驗綜合比較不同毒力NDV感染引起的炎癥反應及S1PR1的表達差異;在此基礎上,研究S1PR1調控NDV引起宿主炎癥反應的分子機制,為臨床上控制NDV感染引起的炎癥反應及應用免疫調節(jié)性藥物防治ND提供理論指導。本研究主要內容如下:1.不同毒力新城疫病毒感染引起的雞炎癥反應及雞S1PR1基因的表達本研究首先選取2株生物學特性差異較大的NDV毒株GM株和La Sota株感染SPF雞,觀察不同毒株引起的炎癥表現,并通過熒光定量PCR對促炎性細胞因子IL-1β的轉錄進行分析。強毒株GM株感染后表現出滲出性炎癥癥狀,感染雞呼吸道出現大量黏液,消化道有明顯的出血點;而La Sota株僅表現出輕微的呼吸道癥狀。組織切片觀察可見,在GM株感染雞的腦、肺臟、脾臟和腺胃中出現出血和大量炎性細胞浸潤等炎性顯微病理變化,La Sota株僅有少量炎性細胞聚集。GM株在小腸、腦、法氏囊、腺胃和盲腸扁桃體中增殖顯著上調,依次為27.5倍、14倍、5.6倍、3.8倍和3.5倍;La Sota株僅在腦中增殖水平較高(6.8倍)。在感染組臟器中檢測到IL-1β的上調表達,GM組高達8.7倍,La Sota組達到6.4倍,GM組的表達水平達顯著高于La Sota組。NDV感染后S1PR1在腦、肝臟、腺胃和法氏囊中表達量較高,最高可達6倍(腦),最低為2倍(法氏囊);在腎臟、小腸和胰腺等組織中表達下調。為研究雞S1PR1的功能,本研究從DF-1細胞中擴增了雞S1PR1基因,并構建了真核表達載體p CI-S1PR1,在DF-1細胞中雞S1PR1基因得到了有效的表達。2.S1PR1參與調控NDV誘導的炎癥反應為探究S1PR1與NDV炎癥的關系,本研究檢測了NDV感染后DF-1細胞中炎性細胞因子及S1PR1的表達:GM感染后IL-1β、IL-6和IL-18表達顯著上調,依次達22倍、41倍和5.5倍,La Sota株感染組與對照組無顯著差異,僅有2倍、1.2倍和1.5倍上調;GM感染誘導IL-8和CCL5 48倍和9.2倍的上調表達,La Sota株感染組也有8.5倍和4.3倍上調表達,顯著高于對照組;TNF-α在La Sota組后呈2.8倍的上調表達,而GM組表達下調。GM株感染引起S1PR1基因2.5倍的上調表達,而La Sota株感染后S1PR1的表達水平無顯著差異;NDV感染3 h起,可在感染細胞中檢測到S1PR1的蛋白表達,且GM組表達水平高于La Sota組。使用W146抑制S1PR1基因表達后,顯著下調IL-6(40倍)和IL-1β(10倍)的轉錄和160 pg/m L、250 pg/m L蛋白表達。S1PR1過表達顯著上調NDV誘導的IL-1β的基因轉錄(5倍)和蛋白表達水平(250 pg/m L),下調IL-6的表達210 pg/m L。抑制或過表達S1PR1基因不影響NDV的復制與增殖。3.S1PR1調控新城疫病毒感染引起炎性反應的信號通路研究為進一步研究S1PR1對NDV炎癥反應的調控機制,選擇與誘導IL-1β表達相關的核轉錄因子NF-κB開展研究。本研究利用NDV強毒GM株感染DF-1細胞,在GM株感染早期Rel A/p65的表達水平顯著上調,在感染3 h其m RNA水平達到6.7倍,至感染6 h達到最高峰(10.8倍),后呈下降趨勢,感染24 h其表達水平與未感染組無顯著差異;NDV感染后細胞熒光素酶值顯著上調,高于對照組12倍;在感染3 h其蛋白表達水平最高,隨著感染時間的延長逐漸降低。W146處理后,NDV感染引起的NF-κB轉錄顯著下調,比DMSO處理組降低了10倍;其熒光素酶值顯著下調了1.5倍。此外,W146處理組NF-κB的蛋白表達水平和亞細胞定位情況與DMSO處理組無顯著差異。由于NF-κB的激活還需要IκB蛋白的磷酸化等過程,因此,本研究對于S1PR1能否調控NF-κB信號通路還不能提供明確的結論,具體通路有待研究。4.不同毒力NDV誘導的雞DC細胞炎癥反應為了更加全面地研究NDV感染引起的雞炎癥反應,本研究選擇具有抗原遞呈功能的樹突狀細胞(DC)作為模型,研究NDV在其中的炎性反應。首先從雛雞骨髓中分離單核細胞,利用GM-CSF和IL-4在體外誘導分化成DC。根據細胞分化周期和病毒感染特性,在細胞培養(yǎng)第3天分別接種NDV毒株GM株和La Sota株,檢測NDV復制以及炎性細胞因和S1PR1的表達。結果顯示,NDV在DC中能夠有效復制并形成細胞病變,GM株最高滴度可達107.2 TCID50/100μL,與La Sota株最高峰值差約為10 000倍。GM組感染后IL-1β(10.2倍)、IFN-β(7.6倍)和CCL5(35.4倍)的表達顯著上調;而La Sota株誘導IL-10表達(峰值為7.5倍),上調CCL5表達(14.3倍)。S1PR1基因在DC細胞中呈下調表達。本研究從NDV感染過程中機體免疫和病毒感染的動態(tài)變化出發(fā),結合對免疫調節(jié)分子S1PR1的研究,對NDV感染導致機體滲出性炎癥的分子機制進行研究。主要發(fā)現了S1PR1分子在NDV感染禽類的炎癥反應中具有調節(jié)細胞因子表達水平的作用,豐富了NDV致病機制的內容,為臨床上控制NDV感染引起滲出性炎癥及應用免疫調節(jié)性藥物防治ND提供科學的理論指導。
[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.
【學位授予單位】：華南農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S852.65

【參考文獻】

相關期刊論文 前1條

1 ;Plasmacytoid Dendritic Cells Act as the Most Competent Cell Type in Linking Antiviral Innate and Adaptive Immune Responses[J];Cellular & Molecular Immunology;2005年06期

，

本文編號：1972125