核酸疫苗改善帕金森病小鼠纹状体多巴胺能神经末梢脱失

发布时间：2018-01-22 07:26

本文关键词： 帕金森病核酸疫苗纹状体免疫治疗　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：探讨优化核酸疫苗改善PD模型小鼠纹状体DA能神经末梢脱失及其可能的机制。方法：提取足量高纯度空质粒（pVAX1）和优化核酸疫苗（pVAX1-IL-4/SYN-B）。将45只正常C57BL/6小鼠（雄性，22-25克）随机分为4组：正常对照组（PBS+PBS，10只）、PBS PD模型组（MPTP+PBS,10只）、空质粒PD模型组(MPTP+pVAX1，10只)和优化核酸疫苗PD模型组（MPTP+pVAX1-IL-4/SYN-B，15只）。后三组小鼠腹腔注射丙磺舒30min后，再向其腹腔注射MPTP建立慢性MPTP PD小鼠模型。而正常对照组小鼠腹腔注射等体积PBS。成功建立PD动物模型后，分别向正常对照组和3组PD组小鼠每侧后肢胫前肌分别注射PBS、PBS、pVAX1、pVAX1-IL-4/SYN-B50μL共100μL，共进行3次免疫。免疫结束后，5只优化核酸疫苗PD模型组小鼠再立体定位于侧脑室注射3μL磷酸氯喹，以同样的方式向剩余各组小鼠注射等体积PBS。对比各组小鼠的行为学变化。通过western blot，免疫组化和免疫荧光检测各组小鼠纹状体区酪氨酸羟化酶（tyrosine hydroxylase，，TH）、突触素（synaptophysin，SYP）、α-突触核蛋白（alpha-synuclein，α-syn）、微管相关蛋白1轻链3（microtubule-associated protein1lightchain3，LC3）、溶酶体相关膜蛋白1（lysosomal-associated membraneprotein1，Lamp1）组织蛋白酶D（cathepsin D，CD）的表达水平。结果： 1.行为学观察在建立慢性MPTP PD小鼠模型的过程中，PD模型组小鼠均先后出现类PD症状：肢体抖动、尾巴上翘，活动缓慢、减少，双后肢站立时后仰等姿势不稳。免疫之后，与正常对照组相比较，PD模型组小鼠运动障碍显著（P＜0.05）。与PBS PD模型组或空质粒PD模型组比较，优化核酸疫苗PD模型组小鼠运动障碍显著改善（P＜0.05）。 2.免疫组化改变免疫组化结果显示：PD模型组小鼠纹状体区TH和SYP表达量较正常对照组明显减少（P＜0.05），优化核酸疫苗PD模型组小鼠纹状体区TH和SYP表达量减少较PBS PD模型组或空质粒PD模型组明显减轻（P＜0.05）。 3. Western blot对比Western blot结果显示：PD模型组小鼠纹状体区TH和SYP表达量较正常对照组明显减少（P＜0.05），优化核酸疫苗PD模型组小鼠纹状体区TH和SYP表达量减少较PBS PD模型组或空质粒PD模型组明显减轻（P＜0.05）。与正常对照组相比较，PD模型组小鼠纹状体区α-syn二聚体明显增多（P＜0.05），CD明显减少（P＜0.05），Lamp1明显减少（P＜0.05）。与PBS PD模型组或空质粒PD模型组相比较，优化核酸疫苗PD模型组小鼠纹状体区α-syn二聚体明显减少（P＜0.05），CD明显增多（P＜0.05），Lamp1明显增多（P＜0.05）。结论：本研究结果表明：慢性MPTP暴露可以损害C57BL/6小鼠运动功能和纹状体DA能神经末梢，分子病理机制可能是溶酶体消耗引起α-syn二聚体增加或二者互为因果。而优化核酸疫苗免疫治疗可以改善慢性MTPT PD模型小鼠运动障碍和纹状体DA能神经末梢脱失，免疫治疗的神经保护机制可能是通过修复溶酶体，促进α-syn二聚体通过自噬-溶酶体途径降解。
[Abstract]:Objective: Objective: to investigate the mechanism of improving the loss of DA nerve endings in striatum of PD model mice by optimizing nucleic acid vaccine. The high purity empty plasmid pVAX1 was extracted and the nucleic acid vaccine pVAX1-IL-4 / SYN-B1 was optimized. 45 normal C57BL / 6 mice (male) were selected. 22-25 g) were randomly divided into 4 groups: 10 PBS PBSs in the normal control group and 10 MPTP PBSs in the PBS PD model group. The empty plasmid PD model group (10 mice) and the optimized nucleic acid vaccine PD model group (MPTP pVAX1-IL-4/SYN-B). 15 mice in the latter three groups were intraperitoneally injected with propanecid for 30 minutes. The model of chronic MPTP PD mice was established by intraperitoneal injection of MPTP, while the normal control group mice were injected intraperitoneally with the same volume of PBSs. After the establishment of PD animal model, the model of PD was successfully established. The normal control group and three groups of PD mice were injected into the anterior tibial muscle of each side of the hind limb, respectively. The PBSS-pVAX1-IL-4 / SYN-B50 渭 L was 100 渭 L. After immunization, 5 mice in PD model group of optimized nucleic acid vaccine were stereoscopically injected with 3 渭 L chloroquine phosphate in lateral ventricle. In the same way, the remaining mice were injected with iso-volume PBSs. The behavioral changes of each group were compared by western blot. The tyrosine hydroxylase (tyrosine hydroxylaseTHN) and synaptophysin (synaptophysin) in the striatum of each group were detected by immunohistochemistry and immunofluorescence. SYP, alpha-synuclein, 伪 -synn. Microtubule-associated protein 1lightchain3 (LC3). Lysosomal-associated membraneprotein1. The expression level of cathepsin DCD in Lamp1. Results: 1.Behavioral observation showed that PD like symptoms occurred successively in the model group of chronic MPTP PD mice: limb jitter, tail upwardness, slow activity and decrease. The postures were unstable when the hind limbs were standing. After immunization, they were compared with the normal control group. The motor dysfunction of PD model group was significantly lower than that of PBS PD model group or blank plasmid PD model group (P < 0.05). In PD model group, the dyskinesia was significantly improved (P < 0.05). 2.Immunohistochemical changes showed that the expression of th and SYP in the striatum of the mice in the weight PD group was significantly lower than that in the normal control group (P < 0.05). The expression of th and SYP in the striatum of PD mice with optimized nucleic acid vaccine was significantly lower than that of PBS PD model or blank plasmid PD model group (P < 0.05). 3. The results of Western blot and Western blot showed that the expression of th and SYP in the striatum of the mice in the group of 10% PD was significantly lower than that in the control group (2). P < 0.05). The expression of th and SYP in the striatum of mice in the PD model group of optimized nucleic acid vaccine was significantly lower than that in the PBS PD model group or the blank plasmid PD model group (P < 0.05), and was significantly lower than that in the normal control group. In PD model group, 伪 -syn dimer in striatum increased significantly (P < 0.05) and CD decreased significantly (P < 0.05). Compared with PBS PD model group or empty plasmid PD model group, Lamp1 decreased significantly (P < 0.05). In PD model group, 伪 -syn dimer in striatum decreased significantly (P < 0.05) and CD increased significantly (P < 0.05). Lamp1 increased significantly (P < 0.05). Conclusion: The results showed that chronic MPTP exposure could damage motor function and striatal DA nerve endings in C57BL / 6 mice. The mechanism of molecular pathology may be the increase of 伪 -syn dimer caused by lysosome consumption or the causality of the two. Optimizing the immunotherapy of nucleic acid vaccine can improve chronic MTPT. PD model mice showed motor disorders and striatal DA nerve endings loss. The neuroprotective mechanism of immunotherapy may be to promote the degradation of 伪 -syn dimer through autophage-lysosome pathway by repairing lysosomes.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.5

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