ALV-J感染后细胞因子变化规律及其促进IL-6产生的机制与效应研究

发布时间：2018-04-27 09:57

本文选题：J亚群禽白血病病毒 + 细胞因子　；参考：《中国农业科学院》2015年硕士论文

【摘要】：J亚群禽白血病(Subgroup J Avian Leukosis)是由J亚群禽白血病病毒(ALV-J)引起的以髓细胞瘤及其他细胞恶性肿瘤为特征的肿瘤性传染性疾病,是禽类重要的免疫抑制病。目前对于其致病性的研究有很多,但都是从病毒本身的特性出发对其增殖能力及致病性做出分析,而忽视了宿主免疫系统在病毒感染过程中发挥的作用。已有研究指出,多种病毒可利用宿主的免疫系统,实现其自身的致病过程。鉴于细胞因子(Cytokine)是免疫原、丝裂原或其他刺激剂诱导细胞产生的低分子量的可溶性蛋白质,具有调节固有免疫和适应性免疫、血细胞生成、细胞生长分化以及修复损伤组织等多种功能,本研究致力于通过检测ALV-J感染后细胞因子表达的变化规律,从宿主免疫系统的角度,探究ALV-J的致瘤机理。本研究首先构建了ALV-J感染的动物模型。在ALV-J感染后的2,3,4,5,6,7,9,12,15,18和21天,取感染组和对照组的外周血淋巴细胞和各主要免疫器官(脾脏、法氏囊和盲肠扁桃体),用双标准曲线荧光定量PCR的方法分别检测其中ALV-J和细胞因子(IL-6,IL-18,IFN-α和IFN-γ)的含量。结果表明ALV-J感染后,随着宿主体内病毒载量的升高,宿主体内细胞因子(IL-6,IL-18,IFN-α和IFN-γ)的表达量也出现逐步增高的现象。当病毒含量达到最高点后突然下降,与此同时,细胞因子的表达量也随之大幅度下降。这个现象说明宿主体内细胞因子的表达与ALV-J的感染密切相关。IL-6是一种重要的多功能促炎性细胞因子,可以调控多种其他细胞因子的表达,在细胞因子的大网络中处于核心地位,是一种重要的免疫物质。已有多个研究指出,IL-6与肿瘤的形成密切相关。鉴于ALV-J是一种重要的免疫抑制性的致肿瘤性疾病,根据IL-6对免疫反应与肿瘤形成的重要作用,本研究随后探究了ALV-J引起IL-6上调表达的机制。实验结果证明,ALV-J可以在体外诱导鸡脾细胞、外周血淋巴细胞及血管内皮细胞中IL-6的表达,与体内实验结果一致。进一步实验结果表明,在体外原代鸡脾细胞中,ALV-J可以通过其p27蛋白促进IL-6的表达,且这种作用呈现出一定的剂量依赖性。同时,细胞信号通路抑制实验证明ALV-J促进IL-6表达的过程需要PI3K和NF-κB细胞信号通路的激活才能实现。随后的Dual-glo Luciferase实验结果显示,当抑制PI3K通路的活性时,ALV-J对NF-κB信号通路的激活作用会明显减弱,表明ALV-J对NF-κB信号通路的激活在一定程度上依赖PI3K细胞信号通路的激活。由此可见,ALV-J可利用其p27蛋白,通过激活PI3K和NF-κB两条细胞信号通路,促进IL-6的表达。为了从机体免疫系统的角度揭示ALV-J的致瘤机理,探究IL-6与ALV-J的致瘤性之间的关系,本研究检测了IL-6在鸡胚体内及体外血管内皮细胞中对血管内皮生长因子(VEGF-A)及其受体(VEGFR-2)表达的影响。结果表明,IL-6在鸡胚体内及体外血管内皮细胞中,均能引起VEGF-A及VEGFR-2的上调表达,且这种效应依赖于由IL-6激活的STAT3细胞信号通路。ALV-J的感染也能够引起VEGF-A及VEGFR-2表达上调,但进一步的RNA干扰实验显示,当干扰IL-6的表达后,ALV-J感染不再能够促进VEGF-A及VEGFR-2的表达。这些结果表明,ALV-J通过促进IL-6的表达,实现其促进VEGF-A及VEGFR-2表达的作用。VEGF-A和VEGFR-2是促进血管内皮细胞增值、增加血管通透性的重要生命物质,对于血管的生成起着至关重要的作用。因此,本研究证明ALV-J通过诱导IL-6的生成而促进VEGF-A和VEGFR-2的表达,促进血管生成,进而有利于其诱导肿瘤的发生。本研究首次从宿主机体免疫系统的角度出发,探究了ALV-J的致瘤机理,揭示了ALV-J病毒利用宿主免疫系统实现自身致病性的致病机制,为ALV-J致病性的研究提供了新的思路,具有重要的意义。
[Abstract]:J subgroup avian leukosis (Subgroup J Avian Leukosis) is a neoplastic infectious disease characterized by myelocytoma and other cell malignancies caused by the J subgroup of avian leukosis virus (ALV-J). It is an important immunosuppressive disease in poultry. There are many studies on its pathogenicity at present, but all of them increase from the characteristics of the virus itself. An analysis of colonization and pathogenicity neglects the role of the host immune system in the process of virus infection. It has been studied that many viruses can use the host's immune system to achieve its own pathogenicity. In view of the cytokine (Cytokine) is the immunogen, the mitogen or other stimulants induce the low molecular weight of the cells. The soluble protein has many functions, such as regulating the innate and adaptive immunity, the generation of blood cells, the cell growth and differentiation, and repairing the damaged tissue. This study is devoted to the detection of the mechanism of ALV-J's tumorigenesis from the angle of the host immune system by detecting the changes in the expression of cytokines after ALV-J infection. The animal model of ALV-J infection. The peripheral blood lymphocytes and the main immune organs (spleen, bursa and cecum tonsil) of the infected and control groups were taken at 2,3,4,5,6,7,9,12,15,18 and 21 days after ALV-J infection, and the ALV-J and cytokine (IL-6, IL-18, IFN- A and IFN- gamma) were detected by double standard curve fluorescence quantitative PCR method. The results showed that after ALV-J infection, the expression of cytokines (IL-6, IL-18, IFN- A and IFN- gamma) in the host increased gradually with the increase of the host virus load in the host. When the virus content reached the highest point, the expression of the cytokine dropped suddenly. The expression of intracellular cytokines in the body is closely related to the infection of ALV-J..IL-6 is an important multi-functional proinflammatory cytokine, which can regulate the expression of a variety of other cytokines. It is an important immune substance in the large network of cytokines. Many studies have shown that IL-6 is closely related to the formation of tumor. In view of the importance of ALV-J as an important immunosuppressive and tumor induced disease, according to the important role of IL-6 in the immunoreaction and tumor formation, this study has subsequently explored the mechanism of ALV-J induced up-regulated expression of IL-6. The experimental results show that ALV-J can induce the expression of IL-6 in the chicken spleen cells, peripheral blood lymphocytes and vascular endothelial cells in vitro. It is consistent with the experimental results in vivo. Further experimental results show that ALV-J can promote the expression of IL-6 through its p27 protein in primary chicken splenocytes in vitro, and this effect presents a certain dose dependence. At the same time, cell signaling pathway inhibition experiments show that the process of ALV-J promoting IL-6 expression requires PI3K and NF- kappa B cell signaling pathways. The subsequent Dual-glo Luciferase experiment showed that when the activity of the PI3K pathway was suppressed, the activation of ALV-J to the NF- kappa B signaling pathway was significantly weakened, indicating that the activation of the NF- kappa B signaling pathway to a certain extent depends on the activation of the PI3K cell signaling pathway. Thus, ALV-J can be used for the use of its p27 protein. Activating the two cell signaling pathways of PI3K and NF- kappa B to promote the expression of IL-6. In order to reveal the mechanism of ALV-J's tumorigenesis from the angle of the immune system of the body and explore the relationship between IL-6 and the tumorigenicity of ALV-J, this study detected the expression of vascular endothelial growth factor (VEGF-A) and its receptor (VEGFR-2) in the vascular endothelial cells of chicken embryos and in vitro. The results showed that IL-6 could induce the up-regulated expression of VEGF-A and VEGFR-2 in both the chicken embryo and in vitro vascular endothelial cells, and this effect depended on the.ALV-J activation of the STAT3 cell signaling pathway activated by IL-6 and the up regulation of VEGF-A and VEGFR-2 expression, but further RNA interference experiments showed that the interference of IL-6 was shown. After that, ALV-J infection no longer promotes the expression of VEGF-A and VEGFR-2. These results suggest that ALV-J promotes the expression of VEGF-A and VEGFR-2 by promoting the expression of IL-6 and VEGFR-2 is an important life substance to promote vascular endothelial cell increment and increase vascular permeability, and plays a vital role in the formation of blood vessels. Therefore, this study proves that ALV-J promotes the expression of VEGF-A and VEGFR-2 by inducing the formation of IL-6 and promotes angiogenesis, which is beneficial to its induction of tumor occurrence. This study is the first time to explore the mechanism of ALV-J's tumorigenesis from the angle of host immune system, and reveals that ALV-J virus can make use of the host immune system to realize its own cause. The pathogenic mechanism of disease provides a new way of thinking for the pathogenicity of ALV-J and is of great significance.

【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3

【参考文献】