REV感染对SPF雏鸡免疫器官免疫功能及Cyclin D1表达的影响
发布时间：2019-06-13 13:13
【摘要】：禽网状内皮增生病(RE)是禽类重要的免疫抑制性致瘤性疾病之一,但由于其临诊症状不典型,该病长期以来没有得到足够重视,现今我国养禽业对禽网状内皮增生病(RE)的预防和防控已经逐渐重视起来,该病由禽网状内皮组织增生病病毒(Reticuloendotheliosis virus,REV)引起。细胞周期素D1(Cyclin D1)是一类与细胞周期功能状态密切相关的蛋白质家族,通过与特定蛋白激酶结合并激活其活性在调节细胞周期G1期到S期中起关键作用。本研究以1日龄SPF雏鸡为研究对象,在成功制备出兔抗鸡Cyclin D1多克隆抗体的基础上,应用流式细胞术检测其免疫器官中淋巴细胞细胞周期分布及CD4+、CD8+T淋巴细胞数量及比值变化、应用细胞培养结合MTT法检测免疫器官中T、B淋巴细胞增殖功能变化、应用Real Time PCR技术检测免疫器官中Cyclin D1 m RNA表达的变化、应用间接ELISA法检测Cyclin D1蛋白含量变化。通过上述各项被检指标的检测,较全面系统的研究了REV感染对SPF雏鸡免疫器官的免疫功能及Cyclin D1表达的影响,为RE防治提供方向。研究结果如下:(1)1日龄SPF雏鸡感染REV后,其免疫器官胸腺和脾脏中,T淋巴细胞增殖功能于REV感染后第1-49 d均不同程度地低于对照雏鸡,两者于病毒感染后第14-28 d与对照雏鸡比较均极显著降低(P0.01);法氏囊未见统计学差异。胸腺CD4+T淋巴细胞数量在REV感染后14-28 d与对照雏鸡相比显著(P0.05)或极显著降低(P0.01)。CD8+T淋巴细胞数量于REV感染后21-35 d极显著低于对照雏鸡(P0.01);在脾脏中CD4+T淋巴细胞数量于病毒感染后7-35 d显著(P0.05)或极显著(P0.01)低于对照雏鸡。CD8+T淋巴细胞数量在REV感染后第3 d、第14 d及第28 d与对照雏鸡相比极显著降低(P0.01);法氏囊CD4+T淋巴细胞数量于病毒感染后3-14 d和第28 d与对照雏鸡相比极显著降低(P0.01)。CD8+T淋巴细胞数量于病毒感染后第1 d极显著低于对照雏鸡(P0.01),第7 d显著高于对照雏鸡(P0.05),35-49 d极显著高于对照雏鸡(P0.01)。胸腺、脾脏、法氏囊CD4+/CD8+T淋巴细胞比值于病毒感染后与对照雏鸡比较出现不同程度的降低(P0.01或P0.05)。表明REV感染1日龄SPF雏鸡后,其主要免疫器官(胸腺、脾脏、法氏囊)细胞免疫功能呈现不同程度的抑制。(2)1日龄SPF雏鸡感染REV后,法氏囊B淋巴细胞增殖功能于病毒感染后14-35 d极显著低于对照雏鸡(P0.01),其余未见统计学差异(P0.05)。脾脏B淋巴细胞增殖功能在病毒感染后14-49 d显著(P0.05)或极显著(P0.01)下降,其余未见统计学差异(P0.05)。表明1日龄SPF雏鸡感染REV后,其法氏囊等免疫器官的体液免疫功能也不同程度下降。(3)REV感染1日龄SPF雏鸡后14-28 d,其胸腺、脾脏、法氏囊淋巴细胞中G0/G1期和S期细胞数占细胞总数的比例与对照雏鸡比较不同程度增高,细胞主要集中在S期中进行DNA复制,G2/M期细胞数比例少。表明REV感染1日龄雏鸡后,其免疫器官中淋巴细胞细胞周期发生变化,S期细胞比例增多,G2/M期的细胞数比例较少,细胞大量停滞于S期,表明细胞增殖受到抑制。(4)REV感染1日龄SPF雏鸡后,其胸腺中Cyclin D1 m RNA表达量不同程度高于对照雏鸡。其中,Cyclin D1 mRNA表达量于REV感染后14-28 d显著(P0.05)或极显著(P0.01)高于对照雏鸡;Cyclin D1蛋白含量在REV感染后第7 d较对照雏鸡极显著降低(P0.01),第14 d较对照组雏鸡显著降低(P0.05),在第35 d显著高于对照雏鸡(P0.05),但未见统计学差异。脾脏中Cyclin D1 m RNA表达量于REV感染后14-21 d极显著(P0.01)高于对照雏鸡,第28 d显著(P0.05)高于对照雏鸡,其余未见统计学差异(P0.05);Cyclin D1蛋白含量在病毒感染后第1 d和第49 d与对照雏鸡相比显著增高(P0.05),第7 d及第21-35d极显著增高(P0.01)。法氏囊中Cyclin D1 mRNA表达量于病毒感染后第21-28 d显著高于对照雏鸡(P0.05);Cyclin D1蛋白含量在病毒感染后第21 d显著增高(P0.05),第14 d、第28 d及第49 d极显著增高(P0.01)。表明SPF雏鸡免疫器官Cyclin D1 mRNA表达及其蛋白含量增多与REV的致病过程及细胞周期分布密切相关。
[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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31
本文编号：2498552
[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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31
【参考文献】
相关期刊论文 前10条
1 袁东方;武莉萍;吴冬梅;杨留勤;杨万才;;Rab3D对食管鳞状细胞癌细胞系增殖及细胞周期素D1表达的影响[J];新乡医学院学报;2016年05期
2 缪佶;鲍衍清;邵红霞;钱琨;秦爱建;;多重实时荧光PCR检测禽网状内皮组织增生症病毒方法的建立以及初步应用[J];中国动物传染病学报;2015年01期
3 邓小芸;祁小乐;李凯;胡峰;高玉龙;高宏雷;王笑梅;;禽网状内皮组织增生病流行现状及防控[J];中国家禽;2015年01期
4 陈雪锋;李恩扩;王肖yN;陈静;胡敬东;;SPF雏鸡感染REV后IL-6和IFN-γ mRNA表达的动态变化[J];中国兽医杂志;2013年06期
5 李恩扩;陈雪锋;孔斌;胡敬东;;SPF雏鸡感染REV后IL-2 mRNA表达的动态变化[J];中国预防兽医学报;2013年01期
6 高玉龙;秦立廷;王笑梅;;家禽病毒性免疫抑制病流行特点与防控对策[J];中国家禽;2012年15期
7 陈吉;崔宏;高美丽;崔琴;;Livin和CyclinD1在慢性萎缩性胃炎伴肠化黏膜中的表达及意义[J];世界华人消化杂志;2012年15期
8 蒋恒波;杨红霞;莫龙;;番茄红素上调细胞周期素依赖性激酶4和细胞周期素D1的表达促进人外周血内皮祖细胞周期进展[J];中国动脉硬化杂志;2011年12期
9 孙洪磊;车国喜;秦梅;杨凤;李宏民;刘思当;;整合禽网状内皮组织增殖病病毒基因的鸡痘病毒野毒株致病性研究[J];中国兽医学报;2011年11期
10 张丹俊;戴银;赵瑞宏;胡晓苗;潘孝成;朱传明;夏松林;姚尚祥;刘鹏义;;安徽省地方品种鸡网状内皮组织增生症血清学调查[J];中国家禽;2011年17期
相关博士学位论文 前4条
1 胡峰;宿主波形蛋白在禽网状内皮组织增生病病毒感染中的作用机制研究[D];中国农业科学院;2015年
2 李凯;禽网状内皮组织增生病基因工程亚单位疫苗及DNA疫苗的研制[D];中国农业科学院;2014年
3 罗利琼;Cyclin D1a/b对肿瘤细胞增殖和转移的影响及其分子机制的研究[D];华中科技大学;2008年
4 孙淑红;禽网状内皮组织增生病病毒与J-亚群白血病病毒的致病性及其疫苗研究[D];山东农业大学;2007年
相关硕士学位论文 前10条
1 崔宏伟;REV感染对SPF雏鸡外周血液及免疫器官TGF-β1含量变化的影响[D];东北农业大学;2016年
2 侯宁;REV感染对SPF雏鸡免疫器官细胞凋亡及Bcl-2、C-myc基因表达的影响[D];东北农业大学;2016年
3 刘文超;REV感染对雏鸡血液主要类型细胞及CyclinD1、p27 mRNA表达的影响[D];东北农业大学;2016年
4 白洁;禽网状内皮组织增殖病病毒及鸡传染性法氏囊病活疫苗对禽流感疫苗免疫效果的影响[D];中国农业科学院;2015年
5 王灵娟;IBDV感染对SPF雏鸡免疫器官TGF_(β1)mRNA表达和免疫功能影响[D];东北农业大学;2013年
6 梁芳;CEF感染REV后部分染色体微卫星不稳定性的研究[D];南京农业大学;2013年
7 陈雪锋;REV感染SPF鸡中IL-6、IL-18和IFN-γ的定量检测[D];山东农业大学;2012年
8 刘溪;吴茱萸碱诱导人肝癌细胞HepG_2细胞周期阻滞机制研究[D];哈尔滨商业大学;2012年
9 张洪海;山东省家禽肿瘤性疾病流行病学调查及GIS预警系统的建立[D];山东农业大学;2009年
10 朱国;禽反转录病毒混合感染肉种鸡抗原定位及诊断的研究[D];山东农业大学;2007年
，本文编号：2498552
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2498552.html
