疫苗猴体神经毒力实验的组织病理学和免疫组织化学评价  

Histopathological and immunohistochemical evaluation of vaccine neurovirulence in monkeys

作　　者：屈哲[1] 杨艳伟[1] 吕建军[1] 李伟[1] 张硕[1] 李萍萍[2] 李珊珊[3] 王雪[1] 汪巨峰[1] 李波[3] 

机构地区：[1]中国食品药品检定研究院国家药物安全评价监测中心药物非临床安全评价研究北京市重点实验室 [2]武汉生物制品研究所有限责任公司 [3]中国食品药品检定研究院

出　　处：《中国新药杂志》2016年第18期2124-2128,共5页Chinese Journal of New Drugs

基　　金：国家"重大新药创制"科技重大专项资助项目(2012ZX09302001)

摘　　要：目的:比较研究疫苗猴体神经毒力临床前安全性评价中的组织病理学评价方法和免疫组织化学评价方法,为疫苗神经毒力鉴定提供病理学诊断金标准。方法:恒河猴丘脑和脊髓腰髓段注射给予乙脑减毒活疫苗、麻疹减毒活疫苗。解剖摘取完整的猴脑和脊髓组织。病理制片修取丘脑、腰髓等部位进行连续切片,取一套组织切片进行常规HE染色,光镜下检查组织病理学改变。另取一套组织切片进行免疫组织化学评价,应用市售的抗GFAP抗体标记星形胶质细胞,抗CD68/Iba1抗体标记小胶质细胞,CD3和CD20抗体分别标记围管浸润中的T淋巴细胞和B淋巴细胞,抗Neu N抗体/MAP-2抗体标记神经元。结果:组织病理学评价结果:乙脑减毒活疫苗可见围管浸润炎症反应、神经胶质细胞增多。麻疹减毒活疫苗可见围管浸润炎症反应。疫苗毒株可见围管浸润炎症反应、神经胶质细胞增多和神经元变性坏死。免疫组织化学评价结果:围管浸润炎症反应病灶经CD3和CD20免疫组化检测呈阳性,表明血管周围有T和B淋巴细胞浸润;神经胶质细胞增多病灶经GFAP和CD68免疫组化检测均呈阳性,表明星形胶质细胞和小胶质细胞均增多或聚集;神经元变性坏死病灶经Neu N和MAP-2免疫组化检测呈阴性反应,表明正常神经元结构蛋白受损后特异性染色呈阴性。结论:神经病理学评价是确认神经损伤及其病变可逆性程度的重要手段,也是评价神经毒性的最经典方法。其中常规的组织病理学评价是最有效的全面性评价神经毒性的方法,为了进一步确认神经毒性的细胞特异性和病理机制,应进行神经系统免疫组织化学评价方法。本研究验证了组织病理学和免疫组织化学评价在神经毒力评价中的重要作用。Objective： To comparatively compare histopathological and immunohistochemical evaluation methods for neurovirulence test of vaccine in monkeys,and provide a gold standard of pathological diagnosis for identification of vaccine neurovirulence. Methods： Rhesus monkeys were administrated with Japanese encephalitis attenuated live vaccine and Measles attenuated live vaccine via injections at hypothalamus and spinal cord lumbar segments. The monkeys were necropsied,and the complete brain and spinal cord tissue were removed. The thalamus and spinal cord tissues were continuously sliced,one set of slices was used for histopathological examination after HE staining,and the other set of slices for immunohistochemical evaluation. Astrocytes were labeled withantibody against GFAP,microglial cells with antibody against CD68/Iba1,the T and B lymphocytes in round tube infiltration with antibodies against CD3 and CD20 individually,and neurons with antibody against Neu N/MAP-2.Results： Histopathological evaluation showed round tube infiltration inflammation and increased glial cells in hypothalamus and spinal cord lumbar segments in Japanese encephalitis attenuated live vaccine group; round tube infiltration inflammation in Measles attenuated live vaccine group; and round tube infiltration inflammation,increased glial cells,and neuronal degeneration and necrosis in virus strain group. Immunohistochemical evaluation showed CD3-positive T-cells and CD20-positive B-cells in the lesion of round tube infiltration inflammation,and GFAP-positive astrocytes and CD68-positive microglia in the increased glial cells; lack of Neu N-and MAP2-positive neurons indicated protein damage of the normal neuronal structure. Conclusion： Neuropathological evaluation is an important means for the confirmation of nerve injury and reversible lesion degree,and the classical method for evaluating neurotoxicity. The general histopathological evaluation is the most effective and comprehensive approach,but the immunohistochemical evaluation should be

关 键 词：神经毒力 组织病理学 免疫组织化学 

分 类 号：R99[医药卫生—毒理学]