本文選題：J亞群禽白血病病毒 + 細(xì)胞因子��； 參考：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：J亞群禽白血病(Subgroup J Avian Leukosis)是由J亞群禽白血病病毒(ALV-J)引起的以髓細(xì)胞瘤及其他細(xì)胞惡性腫瘤為特征的腫瘤性傳染性疾病,是禽類重要的免疫抑制病。目前對于其致病性的研究有很多,但都是從病毒本身的特性出發(fā)對其增殖能力及致病性做出分析,而忽視了宿主免疫系統(tǒng)在病毒感染過程中發(fā)揮的作用。已有研究指出,多種病毒可利用宿主的免疫系統(tǒng),實(shí)現(xiàn)其自身的致病過程。鑒于細(xì)胞因子(Cytokine)是免疫原、絲裂原或其他刺激劑誘導(dǎo)細(xì)胞產(chǎn)生的低分子量的可溶性蛋白質(zhì),具有調(diào)節(jié)固有免疫和適應(yīng)性免疫、血細(xì)胞生成、細(xì)胞生長分化以及修復(fù)損傷組織等多種功能,本研究致力于通過檢測ALV-J感染后細(xì)胞因子表達(dá)的變化規(guī)律,從宿主免疫系統(tǒng)的角度,探究ALV-J的致瘤機(jī)理。本研究首先構(gòu)建了ALV-J感染的動物模型。在ALV-J感染后的2,3,4,5,6,7,9,12,15,18和21天,取感染組和對照組的外周血淋巴細(xì)胞和各主要免疫器官(脾臟、法氏囊和盲腸扁桃體),用雙標(biāo)準(zhǔn)曲線熒光定量PCR的方法分別檢測其中ALV-J和細(xì)胞因子(IL-6,IL-18,IFN-α和IFN-γ)的含量。結(jié)果表明ALV-J感染后,隨著宿主體內(nèi)病毒載量的升高,宿主體內(nèi)細(xì)胞因子(IL-6,IL-18,IFN-α和IFN-γ)的表達(dá)量也出現(xiàn)逐步增高的現(xiàn)象。當(dāng)病毒含量達(dá)到最高點(diǎn)后突然下降,與此同時,細(xì)胞因子的表達(dá)量也隨之大幅度下降。這個現(xiàn)象說明宿主體內(nèi)細(xì)胞因子的表達(dá)與ALV-J的感染密切相關(guān)。IL-6是一種重要的多功能促炎性細(xì)胞因子,可以調(diào)控多種其他細(xì)胞因子的表達(dá),在細(xì)胞因子的大網(wǎng)絡(luò)中處于核心地位,是一種重要的免疫物質(zhì)。已有多個研究指出,IL-6與腫瘤的形成密切相關(guān)。鑒于ALV-J是一種重要的免疫抑制性的致腫瘤性疾病,根據(jù)IL-6對免疫反應(yīng)與腫瘤形成的重要作用,本研究隨后探究了ALV-J引起IL-6上調(diào)表達(dá)的機(jī)制。實(shí)驗(yàn)結(jié)果證明,ALV-J可以在體外誘導(dǎo)雞脾細(xì)胞、外周血淋巴細(xì)胞及血管內(nèi)皮細(xì)胞中IL-6的表達(dá),與體內(nèi)實(shí)驗(yàn)結(jié)果一致。進(jìn)一步實(shí)驗(yàn)結(jié)果表明,在體外原代雞脾細(xì)胞中,ALV-J可以通過其p27蛋白促進(jìn)IL-6的表達(dá),且這種作用呈現(xiàn)出一定的劑量依賴性。同時,細(xì)胞信號通路抑制實(shí)驗(yàn)證明ALV-J促進(jìn)IL-6表達(dá)的過程需要PI3K和NF-κB細(xì)胞信號通路的激活才能實(shí)現(xiàn)。隨后的Dual-glo Luciferase實(shí)驗(yàn)結(jié)果顯示,當(dāng)抑制PI3K通路的活性時,ALV-J對NF-κB信號通路的激活作用會明顯減弱,表明ALV-J對NF-κB信號通路的激活在一定程度上依賴PI3K細(xì)胞信號通路的激活。由此可見,ALV-J可利用其p27蛋白,通過激活PI3K和NF-κB兩條細(xì)胞信號通路,促進(jìn)IL-6的表達(dá)。為了從機(jī)體免疫系統(tǒng)的角度揭示ALV-J的致瘤機(jī)理,探究IL-6與ALV-J的致瘤性之間的關(guān)系,本研究檢測了IL-6在雞胚體內(nèi)及體外血管內(nèi)皮細(xì)胞中對血管內(nèi)皮生長因子(VEGF-A)及其受體(VEGFR-2)表達(dá)的影響。結(jié)果表明,IL-6在雞胚體內(nèi)及體外血管內(nèi)皮細(xì)胞中,均能引起VEGF-A及VEGFR-2的上調(diào)表達(dá),且這種效應(yīng)依賴于由IL-6激活的STAT3細(xì)胞信號通路。ALV-J的感染也能夠引起VEGF-A及VEGFR-2表達(dá)上調(diào),但進(jìn)一步的RNA干擾實(shí)驗(yàn)顯示,當(dāng)干擾IL-6的表達(dá)后,ALV-J感染不再能夠促進(jìn)VEGF-A及VEGFR-2的表達(dá)。這些結(jié)果表明,ALV-J通過促進(jìn)IL-6的表達(dá),實(shí)現(xiàn)其促進(jìn)VEGF-A及VEGFR-2表達(dá)的作用。VEGF-A和VEGFR-2是促進(jìn)血管內(nèi)皮細(xì)胞增值、增加血管通透性的重要生命物質(zhì),對于血管的生成起著至關(guān)重要的作用。因此,本研究證明ALV-J通過誘導(dǎo)IL-6的生成而促進(jìn)VEGF-A和VEGFR-2的表達(dá),促進(jìn)血管生成,進(jìn)而有利于其誘導(dǎo)腫瘤的發(fā)生。本研究首次從宿主機(jī)體免疫系統(tǒng)的角度出發(fā),探究了ALV-J的致瘤機(jī)理,揭示了ALV-J病毒利用宿主免疫系統(tǒng)實(shí)現(xiàn)自身致病性的致病機(jī)制,為ALV-J致病性的研究提供了新的思路,具有重要的意義。
[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.

【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

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本文編號：1810264