本文選題：ALV-J + 免疫抑制�。� 參考：《華南農(nóng)業(yè)大學》2015年博士論文

【摘要】：禽白血病是由反轉(zhuǎn)錄病毒科甲型反轉(zhuǎn)錄病毒屬禽反轉(zhuǎn)錄病毒引起的一種禽類多種腫瘤性疾病的統(tǒng)稱。此病自發(fā)現(xiàn)以后就在世界各國雞群中廣泛流行,主要通過垂直傳播并引發(fā)腫瘤產(chǎn)生,導致雞死亡和消瘦、生長發(fā)育不良,同時引起機體抵抗力下降和免疫抑制,是危害養(yǎng)禽業(yè)的主要疾病之一。由于對禽白血病的研究熱點往往都集中在病毒的流行病學,致病性,致瘤和分子結(jié)構(gòu)與功能方面,對于其導致免疫抑制的機制研究的并不十分清楚。本研究通過體外誘導培養(yǎng)免疫細胞分化和發(fā)育成熟的技術(shù),研究和分析了ALV-J在樹突狀細胞分化、發(fā)育和成熟的過程中所造成的影響。由于樹突狀細胞的制作和分化過程研究的比較清楚,與其在抗原遞呈和抗腫瘤方面的特殊地位,我們選擇了建立體外培養(yǎng)并誘導分化的樹突狀細胞培養(yǎng)方法,并以此為基礎,研究ALV-J對其分化成熟的影響。為了研究病毒是否能順利感染分化中不同狀態(tài)的樹突狀細胞,根據(jù)細胞培養(yǎng)和病毒培養(yǎng)所需的時間,在細胞分化培養(yǎng)的第1 d、2 d、4 d、6 d分別接種ALV-J。結(jié)果發(fā)現(xiàn),在第1d、2d、4d病毒可以成功感染細胞,第六天接毒的樣品,病毒感染失敗。在實驗過程中發(fā)現(xiàn)在接毒后的5-6天內(nèi),細胞的生長,分化情況并無明顯差異。在接毒6天以后,細胞分化并接近成熟的時期,細胞數(shù)量開始減少,并且出現(xiàn)了形態(tài)變化。后經(jīng)細胞凋亡實驗發(fā)現(xiàn),ALV-J可以導致細胞在分化的后期接近成熟時,細胞開始過早的凋亡。對部分TLR和細胞因子的mRNA表達水平的檢測結(jié)果也顯示,大部分檢測的mRNA表達水平都出現(xiàn)了顯著性下調(diào)。結(jié)果表明,ALV-J的感染抑制了樹突狀細胞的成熟,并且造成了樹突狀細胞的凋亡,并影響到了細胞的正常生物學功能。為了研究ALV-J對樹突狀細胞生物學功能影響的機制,進一步分析了樹突狀細胞的miRNA表達水平。發(fā)現(xiàn)了122種差異表達的miRNA。其中已知的miRNA81種,未知新發(fā)現(xiàn)的miRNA41種。在已知的81種差異表達的miRNA中,發(fā)現(xiàn)了4種與細胞免疫功能,ALV致病性有關(guān)的miRNA,分別是gga-mir-221、gga-mir-211、gga-mir-204和gga-mir-6651,這些miRNA共同調(diào)控著細胞內(nèi)源性刺激應答、抗原遞呈和加工、細胞凋亡和慢性骨髓白血病相關(guān)的細胞功能和通路。經(jīng)miRNA表達水平的分析,這些細胞功能和代謝通路是呈激活狀態(tài)的。說明,病毒感染沒有直接對細胞的抗原遞呈功能產(chǎn)生影響。同時,發(fā)現(xiàn)這些miRNA與慢性骨髓白血病有關(guān),可能與ALV-J導致骨髓瘤型白血病有一定的關(guān)系。對顯著性差異的細胞功能和代謝通路的分析結(jié)果發(fā)現(xiàn)多集中于細胞的生理代謝和能量物質(zhì)代謝方面。ALV-J的感染使得這些功能的調(diào)控基因發(fā)生紊亂。細胞在分化的過程中,需要多種基因的調(diào)控,也需要營養(yǎng)和能量的供給。而ALV-J感染會影響到調(diào)控基因的表達,進一步影響細胞正常的生理和新陳代謝功能,從而導致細胞分化失敗,活性降低和凋亡等。本研究發(fā)現(xiàn)ALV-J會嚴重影響樹突狀分化成熟。而這種情況可能設計大部分甚至所有免疫細胞的分化和成熟,進而導致免疫抑制。本研究首次從免疫細胞分化的角度去分析研究ALV-J導致免疫抑制的機制,對于ALV-J導致免疫抑制機制的研究,提供了一個新的研究方向,將有助于進一步全面的研究ALV-J導致免疫抑制的原因和機制。
[Abstract]:Avian leukosis is a common name for a variety of fowl diseases caused by the retrovirus of the family antiretroviral family. This disease has been widely popular among chickens in the world since its discovery, mainly through vertical transmission and initiation of cancer, resulting in death and emaciation of chickens, growth and development, and the cause of the body. The decline of resistance and immunosuppression is one of the major diseases that harm the poultry industry. The research focus on avian leukemia is often concentrated on the epidemiology, pathogenicity, tumorigenicity, molecular structure and function of the virus, and the mechanism of its immunosuppression is not very clear. This study has been induced in vitro by induction of immunization. The effects of ALV-J on the differentiation, development and maturation of dendritic cells are studied and analyzed. The study of the process and differentiation of dendritic cells is clear. We choose to establish in vitro culture and induce differentiation in specific sites of antigen presentation and antitumor. In order to study whether the virus could successfully infect different dendritic cells in the differentiation of the virus, in order to investigate whether the virus could successfully infect the dendritic cells of different states of differentiation, the results were found in first D, 2 D, 4 D, 6 D, respectively, in cell differentiation and culture, in order to study the effect of ALV-J on the differentiation and maturation of the cells. 1D, 2D, 4D virus can infect cells successfully, and the virus infection failed in sixth days. During the experiment, there was no obvious difference in the growth and differentiation of cells within 5-6 days after being poisoned. After 6 days of poison, the cell differentiation was close to the mature period, the number of cells began to decrease, and the morphological changes occurred. The apoptosis test found that ALV-J could lead to premature apoptosis when the cells were close to maturity in the late stage of differentiation. The results of mRNA expression levels of partial TLR and cytokines also showed that most of the levels of mRNA expression were significantly downregulated. The results showed that the infection of ALV-J inhibited the formation of dendritic cells. Mature, and cause the apoptosis of dendritic cells, and affect the normal biological function of the cells. In order to study the mechanism of the effect of ALV-J on the biological function of dendritic cells, the miRNA expression level of dendritic cells was further analyzed. The known miRNA81 species in 122 differentially expressed miRNA. were found, and the newly discovered miRNA41 was unknown. Species. In 81 known differentially expressed miRNA, 4 miRNA related to cellular immune function, ALV pathogenicity, are gga-mir-221, gga-mir-211, gga-mir-204 and gga-mir-6651, respectively. These miRNA co regulate the cellular power of cell endogenous stimulus response, antigen presentation and addition, cell apoptosis and chronic myelogenous leukemia. Energy and pathway. Through the analysis of miRNA expression level, these cell functions and metabolic pathways are activated. It shows that the virus infection does not directly affect the antigen presenting function of the cells. At the same time, it is found that these miRNA are associated with chronic myelogenous leukemia and may have a certain relationship with ALV-J induced myeloma leukemia. The analysis of different cellular and metabolic pathways found that the.ALV-J infection in the physiological metabolism of cells and the metabolism of energy materials caused disorder of the regulatory genes of these functions. In the process of differentiation, the cells needed the regulation of many genes and the supply of nutrition and energy. And the ALV-J infection would affect the regulation. The expression of controlled genes further affects the normal physiological and metabolic functions of the cells, which leads to the failure of cell differentiation, reduction of activity and apoptosis. This study found that ALV-J can seriously affect the maturation of dendritic differentiation. This situation may design most of the differentiation and maturation of all immune cells, which may lead to immunosuppression. The study for the first time from the angle of immune cell differentiation to analyze the mechanism of ALV-J induced immunosuppression provides a new direction for the study of the mechanism of ALV-J induced immunosuppression, which will help to further study the causes and mechanisms of ALV-J to lead to immunosuppression.
【學位授予單位】：華南農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：S858.31

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