细胞周素D与禽网状内皮组织增生病病毒免疫致病机制的研究

Avian reticuloendothelial hyperplasia(RE) is not only a kind of neoplastic diseases of poultry, is also an important immunosuppressive disease. Cyclin(Cyc) is a kind of cell cycle related proteins family. Cyclin in many diseases has changed, especially the occurrence of tumor diseases are inseparable. Cyclin and animal tumor disease, especially cyclin and animal viral immunosuppressive tumor diseases, has not been reported to data. The project is based on preliminary, SPF chickens for animals and chick embryo fibroblasts(CEF) of the study, application new theories and new technologies of molecular biology, immunology, and pathology, through the content and its mRNA expression of cyclin D, the number and proliferative capacity of T and B lymphocytes, the number of CD4+ and CD8+ T lymphocytes subsets, the content or the number of IgG, IgM, IgA producing cells, induced activity and mRNA expression of IL-2, TNF, IFN and other cytokines in peripheral blood, thymus, bursa of Fabricius, Harder gland and cecal tonsil of chickens detection, combined with the main the organs and tissues pathological morphology and image analysis. The relativity of the immune pathogenic mechanism of avian reticuloendothelial hyperplasia viral(REV) with cyclins have been studied from cells, subcellular and molecular level and to clarify its detailed mechanism. In order to provide important reference basis for the further study of animal tumor disease development and human viral tumor diseases prevention.

禽网状内皮组织增生病（RE）不仅是禽的一种肿瘤性疾病，也是一种重要的免疫抑制病；细胞周期素（Cyc）是一类与细胞周期相关的蛋白质家族，其变化与许多疾病，尤其是肿瘤性疾病的发生密不可分，Cyc与肿瘤性疾病，特别是与动物病毒性免疫抑制肿瘤病发生的关系，迄今未见报道。本项目通过体内与体外相结合的研究策略，应用分子生物学、细胞培养、免疫病理学等新理论及新技术，经鸡胚成纤维细胞、SPF雏鸡血液及胸腺、脾脏、哈德尔腺等免疫器官组织Cyc D及mRNA表达，T、B细胞数量及功能、CD4+、CD8+细胞亚群和IgG、IgM、IgA含量及其生成细胞等相关免疫指标；IL-2、TNF、IFN等细胞因子诱生活性及mRNA表达的检测；结合主要器官组织病理形态变化及图像分析。从细胞、亚细胞和分子水平研究REV的免疫致病机制及其与Cyc的相关性，为进一步探讨动物肿瘤性疾病的发生及人类病毒性肿瘤病防治提供重要的参考依据。

为探讨网状内皮组织增殖病病毒（REV）对SPF雏鸡的免疫致病作用，并阐明其机制。以SPF雏鸡和鸡胚为研究对象，采取体内体外相结合研究策略，将分子生物学、生物信息学、免疫学、细胞培养等技术与病理学方法有机结合，通过miRNA表达及靶基因mRNA表达、细胞周期素及蛋白、细胞凋亡、REV含量及分布和黏膜免疫相关指标动态变化的检测以及主要器官病理形态变化的观察，对REV感染鸡胚成纤维细胞、SPF雏鸡血液及主要器官组织上述被检指标进行了全面系统检测。研究发现：（1）REV感染雏鸡后第21天，有30个miRNA表达上调，33个下调，差异表达miRNA靶基因Caspase-6，Caspase-7和P27表达也显著升高，而Mcl-1表达明显降低；第28天，可见8个miRNA表达上调，17个下调，差异表达miRNA靶基因CCNA1、CCNB2、CCND3和c-myc表达均不同程度上调，表明REV通过调控miRNA表达提高促凋亡因子mRNA表达，导致免疫器官细胞凋亡，REV感染通过促进细胞周期素蛋白表达，激活原癌基因进而促进肿瘤发生。（2）REV感染所致的雏鸡初期免疫器官淋巴细胞周期阻滞、细胞增殖能力抑制是病毒导致淋巴细胞数量减少及其功能下降的主要因素之一。（3）免疫器官淋巴细胞凋亡引起的淋巴细胞数量减少与REV感染雏鸡免疫功能下降密切相关。（4）免疫器官中Cyclin D1和E 及CDK4和2表达降低或升高参与了REV感染导致雏鸡免疫功能抑制和恢复。（5）免疫器官Cyclin D1 mRNA表达和蛋白含量升高，S期淋巴细胞所占总细胞数比例上升，G2/M期细胞所占比例下降。表明Cyclin D1与REV的致病密切相关。（6）REV感染SPF雏鸡中枢免疫器官的病毒载量明显高于外周免疫器官，表明中枢免疫器官是REV的主要靶器官。该项目为进一步研究REV的免疫致病机制和兽医临诊防治（制）RE提供了重要参考依据。