【摘要】：禽網(wǎng)狀內(nèi)皮增生病(RE)是禽類重要的免疫抑制性致瘤性疾病之一,但由于其臨診癥狀不典型,該病長期以來沒有得到足夠重視,現(xiàn)今我國養(yǎng)禽業(yè)對禽網(wǎng)狀內(nèi)皮增生病(RE)的預(yù)防和防控已經(jīng)逐漸重視起來,該病由禽網(wǎng)狀內(nèi)皮組織增生病病毒(Reticuloendotheliosis virus,REV)引起。細(xì)胞周期素D1(Cyclin D1)是一類與細(xì)胞周期功能狀態(tài)密切相關(guān)的蛋白質(zhì)家族,通過與特定蛋白激酶結(jié)合并激活其活性在調(diào)節(jié)細(xì)胞周期G1期到S期中起關(guān)鍵作用。本研究以1日齡SPF雛雞為研究對象,在成功制備出兔抗雞Cyclin D1多克隆抗體的基礎(chǔ)上,應(yīng)用流式細(xì)胞術(shù)檢測其免疫器官中淋巴細(xì)胞細(xì)胞周期分布及CD4+、CD8+T淋巴細(xì)胞數(shù)量及比值變化、應(yīng)用細(xì)胞培養(yǎng)結(jié)合MTT法檢測免疫器官中T、B淋巴細(xì)胞增殖功能變化、應(yīng)用Real Time PCR技術(shù)檢測免疫器官中Cyclin D1 m RNA表達的變化、應(yīng)用間接ELISA法檢測Cyclin D1蛋白含量變化。通過上述各項被檢指標(biāo)的檢測,較全面系統(tǒng)的研究了REV感染對SPF雛雞免疫器官的免疫功能及Cyclin D1表達的影響,為RE防治提供方向。研究結(jié)果如下:(1)1日齡SPF雛雞感染REV后,其免疫器官胸腺和脾臟中,T淋巴細(xì)胞增殖功能于REV感染后第1-49 d均不同程度地低于對照雛雞,兩者于病毒感染后第14-28 d與對照雛雞比較均極顯著降低(P0.01);法氏囊未見統(tǒng)計學(xué)差異。胸腺CD4+T淋巴細(xì)胞數(shù)量在REV感染后14-28 d與對照雛雞相比顯著(P0.05)或極顯著降低(P0.01)。CD8+T淋巴細(xì)胞數(shù)量于REV感染后21-35 d極顯著低于對照雛雞(P0.01);在脾臟中CD4+T淋巴細(xì)胞數(shù)量于病毒感染后7-35 d顯著(P0.05)或極顯著(P0.01)低于對照雛雞。CD8+T淋巴細(xì)胞數(shù)量在REV感染后第3 d、第14 d及第28 d與對照雛雞相比極顯著降低(P0.01);法氏囊CD4+T淋巴細(xì)胞數(shù)量于病毒感染后3-14 d和第28 d與對照雛雞相比極顯著降低(P0.01)。CD8+T淋巴細(xì)胞數(shù)量于病毒感染后第1 d極顯著低于對照雛雞(P0.01),第7 d顯著高于對照雛雞(P0.05),35-49 d極顯著高于對照雛雞(P0.01)。胸腺、脾臟、法氏囊CD4+/CD8+T淋巴細(xì)胞比值于病毒感染后與對照雛雞比較出現(xiàn)不同程度的降低(P0.01或P0.05)。表明REV感染1日齡SPF雛雞后,其主要免疫器官(胸腺、脾臟、法氏囊)細(xì)胞免疫功能呈現(xiàn)不同程度的抑制。(2)1日齡SPF雛雞感染REV后,法氏囊B淋巴細(xì)胞增殖功能于病毒感染后14-35 d極顯著低于對照雛雞(P0.01),其余未見統(tǒng)計學(xué)差異(P0.05)。脾臟B淋巴細(xì)胞增殖功能在病毒感染后14-49 d顯著(P0.05)或極顯著(P0.01)下降,其余未見統(tǒng)計學(xué)差異(P0.05)。表明1日齡SPF雛雞感染REV后,其法氏囊等免疫器官的體液免疫功能也不同程度下降。(3)REV感染1日齡SPF雛雞后14-28 d,其胸腺、脾臟、法氏囊淋巴細(xì)胞中G0/G1期和S期細(xì)胞數(shù)占細(xì)胞總數(shù)的比例與對照雛雞比較不同程度增高,細(xì)胞主要集中在S期中進行DNA復(fù)制,G2/M期細(xì)胞數(shù)比例少。表明REV感染1日齡雛雞后,其免疫器官中淋巴細(xì)胞細(xì)胞周期發(fā)生變化,S期細(xì)胞比例增多,G2/M期的細(xì)胞數(shù)比例較少,細(xì)胞大量停滯于S期,表明細(xì)胞增殖受到抑制。(4)REV感染1日齡SPF雛雞后,其胸腺中Cyclin D1 m RNA表達量不同程度高于對照雛雞。其中,Cyclin D1 mRNA表達量于REV感染后14-28 d顯著(P0.05)或極顯著(P0.01)高于對照雛雞;Cyclin D1蛋白含量在REV感染后第7 d較對照雛雞極顯著降低(P0.01),第14 d較對照組雛雞顯著降低(P0.05),在第35 d顯著高于對照雛雞(P0.05),但未見統(tǒng)計學(xué)差異。脾臟中Cyclin D1 m RNA表達量于REV感染后14-21 d極顯著(P0.01)高于對照雛雞,第28 d顯著(P0.05)高于對照雛雞,其余未見統(tǒng)計學(xué)差異(P0.05);Cyclin D1蛋白含量在病毒感染后第1 d和第49 d與對照雛雞相比顯著增高(P0.05),第7 d及第21-35d極顯著增高(P0.01)。法氏囊中Cyclin D1 mRNA表達量于病毒感染后第21-28 d顯著高于對照雛雞(P0.05);Cyclin D1蛋白含量在病毒感染后第21 d顯著增高(P0.05),第14 d、第28 d及第49 d極顯著增高(P0.01)。表明SPF雛雞免疫器官Cyclin D1 mRNA表達及其蛋白含量增多與REV的致病過程及細(xì)胞周期分布密切相關(guān)。
[Abstract]:The avian reticuloendotheliosis (RE) is one of the important immunosuppressant-causing diseases of poultry, but because of its atypical symptoms, it has not been paid enough attention for a long time. The prevention and control of avian reticuloendotheliosis (RE) has been paid more and more attention in China. The disease is caused by the virus (REV) of reticuloendotheliosis virus (REV). Cyclin D1 (Cyclin D1) is a family of proteins that are closely related to the cell cycle functional state, and plays a key role in the regulation of the cell cycle G1 phase to the S phase by binding to a specific protein kinase and activating its activity. On the basis of the successful preparation of the anti-chicken Cyclin D1 polyclonal antibody, the cell cycle distribution and the number and ratio of CD4 +, CD8 + T lymphocytes in the immune organs were detected by flow cytometry. Cell culture and MTT were used to detect the changes of T and B lymphocyte proliferation in the immune organs. The changes of the expression of Cyclin D1 mRNA in the immune organs were detected by Real Time PCR, and the content of Cyclin D1 protein was detected by indirect ELISA. The effects of REV infection on the immune function and the expression of Cyclin D1 in the immune organs of the SPF chickens were studied by the detection of the above-mentioned indexes, and the direction of the RE control was provided. The results of the study were as follows: (1) After the infection of REV in 1-day-old SPF chickens, the proliferation of T-lymphocytes in the thymus and spleen of the immune organs was lower than that of the control chicks after REV infection, both in the 14th to 28th day after the virus infection and the control chicks (P0.01). There was no statistical difference in the bursa of Fabrici@@ The number of CD4 + T lymphocytes in the thymus was significantly lower than that of the control (P0.05) or very significantly (P0.01). The number of CD8 + T lymphocytes was significantly lower than that of the control (P0.01). The number of CD4 + T lymphocytes in the spleen was significantly lower than that of the control (P0.05) or very significant (P0.01). The number of CD8 + T lymphocytes was significantly lower in the 3rd day, 14th day and 28th day after REV infection (P0.01). The number of CD4 + T lymphocytes in the bursa of Fabricius was significantly lower than that of the control (P0.01), and the number of CD8 + T lymphocytes was significantly lower than that of the control (P0.01). The 7th day was significantly higher than that of the control (P0.05), and the rate of 35-49d was significantly higher than that of the control (P0.01). The ratio of CD4 +/ CD8 + T-lymphocyte in thymus, spleen and bursa of Fabricius bursa of Fabricius was lower than that of control chicks (P0.01 or P0.05). The immune function of the main immune organs (thymus, spleen and bursa of Fabricius) of the 1-day-old SPF chicks infected with REV was inhibited. (2) After the infection of REV in 1-day-old SPF chicks, the proliferation of the B-lymphocytes in the bursa of Fabricius was significantly lower than that of the control (P0.01), and the rest showed no statistical difference (P0.05). The proliferation of B-lymphocyte in the spleen was significantly higher than that in 14-49d after the virus infection (P0.05) or very significant (P0.01), and there was no statistical difference (P0.05). The humoral immune function of the immune organs, such as the bursa of Fabricius, was also decreased after the infection of REV in SPF chickens of 1 day of age. (3) The ratio of the number of cells in G0/ G1 phase and S-phase cells in the thymus, spleen and bursa of Fabricius of the 1-day-old SPF chicks was higher than that of the control chicks, and the number of cells in the G2/ M phase was less. The cell cycle of the lymphocytes in the immune organs of the 1-day-old chicks infected with REV was changed, the proportion of the S-phase cells increased, the number of cells in the G2/ M phase was less, and the number of the cells remained in the S phase, indicating that the cell proliferation was inhibited. (4) The expression of Cyclin D1 mRNA in the thymus of the 1-day-old SPF chicks infected with REV was higher than that of the control chicks. The expression of Cyclin D1 mRNA was significantly higher than that of control chicks in 14-28d after REV infection (P0.05) or very significant (P0.01). The level of Cyclin D1 protein was significantly lower than that of control chicks after REV infection (P0.01). There was no statistical difference in the 35d significantly higher than that of the control (P0.05). The expression of Cyclin D1 mRNA in the spleen was significantly higher than that of the control (P0.05). The level of Cyclin D1 protein was significantly higher than that of the control (P0.05), the 7th day and the 21st-35d (P0.01). The expression of Cyclin D1 mRNA in the bursa of Fabricius was significantly higher than that of the control (P0.05). The content of Cyclin D1 in the bursa of Fabricius was significantly higher than that of the control (P0.05). The expression of Cyclin D1 mRNA and its protein content in the immune organs of SPF chickens were closely related to the pathogenesis of REV and the distribution of cell cycle.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.31

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