共聚物-1直接免疫MPTP模型小鼠保护多巴胺能神经元

Copolymer-1 Protects Dopaminergic Neurons in An 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Mouse Model of Parkinson’s Disease

目的探讨共聚物-1(copolymer-1,Cop-1)直接免疫对小剂量1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)持续注射C57BL/6J小鼠黑质多巴胺(dopamine,DA)神经元保护作用的影响。方法雄性C57BL/6J小鼠分为MPTP模型组、造模后Cop-1免疫组、造模时Cop-1免疫组、造模前Cop-1免疫组、Cop-1免疫组和正常对照组。MPTP模型组动物仅接受MPTP注射,造模后Cop-1免疫组在MPTP连续注射7d后接受Cop-1免疫;造模时Cop-1免疫组在第1次MPTP注射后立即接受Cop-1免疫;造模前Cop-1免疫组在接受Cop-1免疫7d后开始注射MPTP,Cop-1免疫组仅接受Cop-1抗原免疫,正常对照组动物仅接受0.9%氯化钠注射液注射。MPTP连续注射24d后处死动物,取脑、脾。脑切片酪氨酸羟化酶免疫组化定量分析黑质DA神经元数;高效液相色谱法(high performance liquidchrom atography,HPLC)分析纹状体DA及其代谢产物含量;苏木精-伊红染色法(hematoxylin-eosin staining,HE)观察脾病理学变化;分离造模后Cop-1免疫组、造模时Cop-1免疫组、造模前Cop-1免疫组和Cop-1免疫组动物脾脏并进行Cop-1致敏淋巴细胞培养。结果与正常对照组相比,MPTP模型组、造模后Cop-1免疫组、造模时Cop-1免疫组、造模前Cop-1免疫组动物脾脏HE染色未见明显差异。与Cop-1免疫组相比,造模后Cop-1免疫组、造模时Cop-1免疫组、造模前Cop-1免疫组均出现大量Cop-1致敏淋巴细胞。黑质DA神经元定量分析显示,与正常对照组比较,持续低剂量MPTP注射使模型对照组黑质DA神经元减少了65.13%,造模后Cop-1免疫组、造模时Cop-1免疫组、造模前Cop-1免疫组黑质DA神经元分别减少了31.68%、27.55%和28.29%,各组DA神经元数明显高于模型对照组(P=0.000,P=0.000,P=0.000)。HPLC法测定纹状体内DA及其代谢产物含量结果显示,与正常对照组相比,模型组DA及其代谢产物含量明显减少。造模后Cop-1免疫组、造模时Cop-1免疫组、造模前Cop-1免疫组类似,但均明显高于模型组。结论在C57BL/6J小鼠慢性MPTP模型中,持续低剂量MPTP注射对小鼠的免疫器官脾的破坏不明显,Cop-1直接免疫可有效对抗MPTP毒性,保护黑质-纹状体系统的DA神经元。

Objective To investigate the neuron-protective effects of copolymer-1（Copolymer-1,Cop-1） on dopaminergic neurons in the continuous low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP） induced C57BL/6J mouse model of Parkinson’s disease.Methods The C57BL/6J mice were randomly divided into 6 groups：group 1,MPTP model,group 2,in which MPTP was injected before the Cop-1 immunization,group 3,MPTP was injected immediately after the Cop-1 immunization,group 4,Cop-1 immunization was performed before MPTP injection,group 5,Cop-1 immunization and group 6,normal control group.The mice except for group 5 and 6 received one i.p.injection of MPTP（5 mg.kg-1） every day.After continuous injection for 24 days,all mice were sacrificed after the day of last MPTP injection.The dopaminergic neurons were analyzed quantitatively using immunohistocheministry of tyrosine hydroxylase（TH） and stereological counts,high performance liquid chromatography（HPLC） was used for analysis of striatal dopamine and its metabolites content,HE staining was used for spleen sections.Spleens of the mice of groups 1-4 were separated and Cop-1 sensitized lymphocytes were cultured.Results Compared with normal control group,the animals of,groups 1-4 showed toxins accumulation in the spleen,but HE staining was not significantly different among the groups.Compared with Cop-1 immunization group,there were also a large number of Cop-1 sensitized lymphocytes in other three Cop-1 immunization groups.Stereological counts revealed that MPTP caused a 65.13% loss of TH-positive neurons in substantia nigra（SN） compared with saline controls.Similar results were observed in MPTP-injected mice that received MPTP injection before Cop-1 immunization,immediately after Cop-1 immunization or Cop-1 immunization was performed before MPTP injection.The results of HPLC determination of striatal dopamine and its metabolites showed that,compared with the control group,MPTP model group dopamine and its metabolites were significantly reduced.The levels of dopamine and its metabolites were similar among groups 2-4,but were significantly higher than those of group 1（P0.05）.Conclusion Our data suggest that in the C57BL/6J mouse model,continuous low-dose MPTP injection did not show obvious destructive effects on the spleen,an organ of the immune system.Direct immunization with Cop-1 could be effective against MPTP toxicity,and protect DA neurons in the substantia nigra.