【作者】 施旅娟；

【导师】 张书霞；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2006， 硕士

【摘要】 猪圆环病毒2型(PCV-2)与猪群中发生的多种疾病相关，包括断奶仔猪多系统衰竭综合征(PMWS)、猪皮炎肾病综合征(PDNS)、A2型先天性震颤(CT)等。其中以PMWS最为严重，已经给全世界养猪业带来了很大的经济损失。研究表明PCV-2感染会导致免疫系统损伤，因此了解病毒与免疫系统间的作用对于阐明病毒引起疾病的机理起到至关重要的作用。本研究的目的是从凋亡和细胞因子水平分析PCV-2感染对免疫系统的影响。 实验中我们选择了9头32日龄PCV-2抗体阴性的普通断奶仔猪。随机分为三组，即空白对照组、PCV-2攻毒组、钥匙孔血蓝蛋白(KLH)刺激组(PCV-2+KLH)，每组3头。感染后32天采集淋巴结、脾脏、心脏、肺脏、肝、肾等器官制作石蜡切片。显微镜下观察可见到PCV-2攻毒组和PCV-2+KLH组猪的主要病变出现在淋巴结、脾脏、肺脏；淋巴结皮质和副皮质区淋巴细胞严重缺失，淋巴小结萎缩，上皮样巨噬细胞浸润，巨噬细胞胞浆内有病毒包涵体；脾脏动脉周围淋巴鞘内巨噬细胞增多；肺泡间隔增厚，出现以巨噬细胞浸润为主的间质性肺炎病变。PCV-2+KLH组仔猪较PCV-2组仔猪病变严重。 病理学变化可见PCV-2对淋巴结的损害最大。为了了解PCV-2对淋巴结的致病机理，我们采集对照组、PCV-2组、钥匙孔血蓝蛋白刺激组(PCV-2+KLH)的腹股沟浅淋巴结，采用免疫组化方法对病毒抗原进行定位，并用末端标记法(TUNEL)和流式细胞法对细胞凋亡进行了定位和定量测定。结果表明，PCV-2感染仔猪后32天，病毒主要存在淋巴小结的上皮样巨噬细胞中；PCV-2组和PCV-2+KLH组仔猪淋巴结中的淋巴细胞凋亡严重，且主要是淋巴小结内的淋巴细胞发生凋亡，流式细胞仪检测PCV-2组和PCV-2+KLH组仔猪淋巴结中的细胞凋亡率分别为3.34％、4.88％，明显高于对照组0.43％的凋亡率。 IL-2和TNF-α主要分别由淋巴细胞和巨噬细胞分泌，在免疫系统中发挥着重要和复杂的作用。本实验用放免的方法对感染PCV-2仔猪血清中IL-2和TNF-α的含量进行动态检测。结果表明，PCV-2感染后仔猪血清中IL-2的变化与对照组相比变化不明显，TNF-α的变化趋势则和对照组完全相反。 综上所述，PCV-2感染后仔猪的主要病变出现在淋巴结、脾脏和肺脏。淋巴结的特征性变化是淋巴小结萎缩和上皮样巨噬细胞浸润。病毒定位和细胞凋亡的研究表更多还原

【Abstract】 Some kinds of diseases are related with porcine circovirus type 2 (PCV-2) in porcine groups including PMWS, PDNS, CT, et al. Especially, PMWS is the most serious and has made great economic losses in the world. As reported previously, PCV -2 may induces the damnification of the immune system. Discerning how PCV-2 interact with the immune system is crucial for a better understanding of viral pathogenesis. The objective of these studies is to explore the effects of PCV-2 infection on immune system at the apoptosis and cytokines.In this report, nine 32-day-old conventional piglets free of PCV-2 antibody were used, distributed in three groups. Three pigs received PCV-2 alone (PCV-2 group). Three pigs were stimulated with KLH after inoculating PCV-2 (PCV-2 + KLH group ) . Three control pigs received nothing (control group). The lymph nodes、 spleens、 hearts、 lungs、 livers and kidneys of these pigs were collected after 32 days postinoculation. The characteristic histopathological lesion have been seen in superficial inguinal lymph nodes, spleens and lungs of the piglets of PCV-2 group and PCV-2 + KLH group: Lymphocyte depletion in cortex and paracortex of lymph node, lymphoid nodule atrophy, epithelioid macrophages infiltration, mtracytoplasmic inclusion bodies in epithelioid macrophages are present in lymph nodes. There is appearance of infiltration with epithelioid macrophages in periarteriolar lymphoid sheath of spleen. Alveolar septum, infiltration with macrophages, is thickened. Histopathological lesions of the piglets of PCV-2 + KLH group were more severe than that of PCV-2 group .Microscopic lesions showed that PCV-2 did a lot of damage to the lymph node. In this study we investigated the pathogenetic mechanism of induction lymphocyte depletion in pig’s lymph nodes infected by PCV-2. The superficial inguinal lymph nodes of pigs in the 3 groups were collected at 32 days post-inoculation (DPI) PCV-2. PCV-2 in the lymph nodes was located with immunohistochemistry(IHC) and the apoptosis were observed by both TUNEL and flow cytometry(FCM). The results indicated that PCV-2 antigens were mainly found in the epithelioid macrophages of lymphoid nodule of pigs on PCV-2 group and PCV-2+KLH group. TUNEL-positive cells were mostly lymphocytes in lymphoid nodule of the above two groups The average apoptotic rates of lymph nodes were 3.34 % and 4.88 % in pigs of PCV-2 group and PCV-2+KLH group by flow cytometry respectively. The data更多还原