实验性自身免疫性脑脊髓炎小鼠模型的构建

Establishment of a Mouse Model of Experimental Autoimmune Encephalomyelitis

目的 通过髓鞘少突胶质细胞糖蛋白(MOG_(35-55))多肽两次免疫C57BL/6J小鼠,构建实验性自身免疫性脑脊髓炎(EAE)模型。方法 将8周龄C57BL/6J雌性小鼠按随机数字表法分为EAE组和对照组,每组10只。EAE组采用MOG_(35-55)多肽与完全弗式佐剂混合后的抗原乳剂,于小鼠双侧腹股沟和后肢分4点皮下注射,首次免疫后6 d注射相同剂量的抗原乳剂加强免疫,并且于首次免疫后0、48 h腹腔注射百日咳毒素;对照组采用不含MOG_(35-55)多肽的CFA,其余方法同EAE组。观察两组小鼠的行为学表现并进行神经功能评分;免疫后第19天,取两组小鼠的脑和脊髓组织进行Black Gold髓鞘染色和髓鞘碱性蛋白(MBP)免疫荧光染色,检测其脱髓鞘情况;小胶质细胞特异性标志物(Iba-1)免疫荧光染色检测两组脑和脊髓组织中小胶质细胞的增殖活化情况。结果 EAE组小鼠在首次免疫后第12天左右开始发病,第19天症状达到高峰,随后开始缓解,发病率为100%,而对照组小鼠只在首次免疫后第14天出现短暂的尾巴无力症状。Black Gold髓鞘染色和MBP免疫荧光染色显示,EAE组小鼠脑和脊髓均出现脱髓鞘;Iba-1免疫荧光染色显示,EAE组小鼠脑和脊髓均出现小胶质细胞增殖活化;而对照组小鼠脑和脊髓并未出现脱髓鞘和小胶质细胞的增殖活化。结论 MOG_(35-55)两次免疫C57BL/6J小鼠,成功构建发病率高的EAE模型,为多发性硬化症的致病机制研究和药物开发奠定基础。

Objective To establish an experimental autoimmune encephalomyelitis(EAE)model in C57BL/6J mice by twice immunizations with myelin oligodendrocyte glycoprotein peptide(MOG35-55).Methods Eight-week-old female C57BL/6J mice were divided into two groups by using a random number table,namely EAE group(n=10)and control group(n=10).For EAE induction,mice were subcutaneously injected with MOG35-55 peptide emulsified in complete Freund's adjuvant(CFA)at four sites on each side of the inguinal groove and posterior limb.Six days after the initial immunization,mice were boosted with the same dose of antigen/adjuvant emulsions.Moreover,pertussis toxin was given intraperitoneally on the day of initial immunization and 48 hours afterwards.The control group received CFA without MOG35-55 and pertussis toxin.All mice were observed and scored daily based on their behavioral changes.On day 19 post-immunization,the brain and spinal cord were removed and examined for demyelination by Black Gold II staining and myelin basic protein(MBP)immunofluorescent staining.The microglial proliferation and activation in the brain and spinal cord were detected by microglia-specific marker(Iba-1)immunofluorescent staining.Results In mice of EAE group,clinical signs of EAE started on day 12 and peaked on day 19 post-immunization,and then subsided gradually,with the incidence of 100%.However,mice of control group only displayed temporary symptoms of tail atony on day 14 post-immunization.Black Gold II staining and MBP immunofluorescence staining showed both brain and spinal cord demyelination in mice of EAE group.Iba-1 immunofluorescence staining showed that microglial proliferation and activation also occurred in the brain and spinal cord of EAE mice.However,no demyelination or microglial proliferation and activation were found in either brain or spinal cord of control group.Conclusion The EAE model with high incidence is successfully established in C57BL/6J mice by twice immunizations with MOG35-55,which lays a foundation for future pathogenesis research and drug discovery of multiple sclerosis(MS).