神经毒力体外评价方法的建立及减毒活疫苗的体外评价  

Development of in vitro evaluation method for neurovirulence and in vitro evaluation of live attenuated vaccines

作　　者：屈哲[1] 张硕[1] 吕建军[1] 霍艳[1] 王雪[1] 袁力勇[2] 马霄[2] 李波[2] QU Zhe ZHANG Shuo LV Jian-jun HUO Yan WANG Xue YUAN Li-yong MA Xiao LI Bo(Beijing Key Laboratory, National Center for Safety Evaluation of Drugs, National Institutes for Food and Drug Control, Beijing 100176, China)

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,北京100176 [2]中国食品药品检定研究院,北京100050

出　　处：《中国生物制品学杂志》2017年第9期963-968,共6页Chinese Journal of Biologicals

基　　金：重大新药创制科技重大专项2015年定向择优课题"生物大分子药物特殊评价关键技术研究(2015ZX09501007-004)

摘　　要：目的通过构建原代大鼠神经细胞混合培养模型,体外评价减毒活疫苗的神经毒力作用。方法取1~3日龄SD大鼠大脑组织,无菌分离获得神经细胞,于DMEM/F12培养基5%CO2培养箱培养7~9 d至混合培养体系稳定。Anti-MAP2、Anti-GFAP抗体免疫细胞技术鉴定神经元、星形胶质细胞。细胞分组:空白对照组,高、中、低剂量非疫苗对照组(β-淀粉酶和长春新碱)、麻疹减毒活疫苗组、腮腺炎减毒活疫苗组、吸附无细胞百白破灭活脊髓灰质炎和b型流感嗜血杆菌(结合)联合疫苗(简称联合疫苗)组。CCK8增殖毒性试验检测细胞活力;免疫荧光标记GFAP、MAP2观察神经细胞形态学变化;ELISA法检测细胞培养上清中促炎性细胞因子TNFα、IL-1β、IL-6浓度。结果原代神经细胞混合培养体系由约50%GFAP(+)星形胶质细胞和约50%MAP-2(+)神经元组成。联合疫苗、长春新碱给药各时间点存在剂量效应关系,麻疹疫苗、腮腺炎疫苗未见细胞毒性。联合疫苗、长春新碱各剂量组神经细胞存活率降低,神经细胞胞体变形,核浆比增大;麻疹疫苗、腮腺炎疫苗各剂量组神经细胞密度和形态未见异常;β-淀粉酶各剂量组未见细胞毒性和形态学改变。联合疫苗和β-淀粉酶组神经细胞培养上清中IL-1β、TNFα浓度显著升高,麻疹疫苗组IL-6浓度显著升高。结论构建了原代大鼠神经细胞培养模型,可用于体外评价减毒活疫苗的神经毒力作用。麻疹和腮腺炎减毒活疫苗未见神经细胞毒性作用。联合疫苗、麻疹疫苗和β-淀粉酶作用于神经细胞与潜在的神经炎症反应发生相关。长春新碱可见神经细胞毒性作用,但未见促炎性细胞因子IL-1β、TNFα、IL-6的显著性改变。Objective To evaluate the neurovirulence of live attenuated vaccines in vitro by construction of mixed culture model of primary rat nerve cells. Methods Nerve cells were isolated aseptically from the brain tissue of SD rats at ages of 1 ~ 3 d, and cultured in DMEM/F12 medium in a 5% carbon dioxide incubator for 7 ~ 9 d until the culture system was stable. Neurons and astrocytes were identified by immunocytochemistry with MAP2 and GFAP antibodies. The cells were divided into blank control, non-vaccine control（amyloid β and vincristine）, live attenuated measles vaccine, live attenuated mumps vaccine as well as acellular DTP, inactivated poliomyelitis and Haemophilus influenzae type b conjugate vaccine（conjugate vaccine for short） groups. The viability of nerve cells was determined by CCK8 proliferation toxicity test, while the morphological changes were observed by IFA with MAP2 and GFAP antibodies. The concentrations of proinflammatory cytokines TNFα, IL-1βand IL-6 in cell culture supernatant were determined by ELISA. Results The mixed culture system of primary nerve cells was composed of approximately 50% MAP-2（＋）neurons and 50% GFAP（＋）astrocytes. Dose-response relationships were observed in the neurotoxicity at various time points in conjugate vaccine and vincristine groups. However, no neurotoxicity was observed in measles vaccine group or mumps vaccine group. Decreased cell survival rate, deformed cell body and increased nuclear-cytoplasmic ratio were observed in conjugate vaccine group and vincristine groups, while no abnormal density or morphology of nerve cells in measles vaccine and mumps vaccine groups. No changes of cytotoxicity and morphology were found in amyloid β group. The IL-1β and TNFα concentrations in nerve cell culture supernatant in conjugate vaccine and amyloid β groups as well as IL-6 concentration in measles vaccine group increased significantly. Conclusion A rat nerve cell culture model was established, which might be used for in vitro evaluation of neurovirulenc

关 键 词：神经毒力 体外评价 减毒活疫苗 神经细胞培养 

分 类 号：R392[医药卫生—免疫学]