雷帕霉素对亚急性MPTP小鼠PD模型P-4EBP1和a-synuclein表达的影响

发布时间：2019-03-11 17:17

【摘要】：目的：（1）探讨亚急性MPTP模型小鼠中P-4EBP1及与a-synuclein表达之间的关系。（2）探讨TOR抑制剂雷帕霉素是否通过P-4EBP1及TOR信号通路影响亚急性MPTP模型小鼠a-synuclein表达。方法：本研究采用亚急性MPTP帕金森病（Parkinson's Disease，PD）小鼠（C57/BL）模型，将48只小鼠按完全随机分组的方法分为：①预处理模型组（Rapamycin and MPTP，Rap+MPTP）、②模型组（Medium and MPTP，Medi+MPTP）、③预处理对照组（Rapamycin andSaline，Rap+Sali）、④空白对照组(Medium and Saline，Medi+Sali)。在同等条件下饲养：①预处理模型组小鼠每日同一时间点注射雷帕霉素(7.5mg/kg)*7d，第3-7日，介质注射后30min时腹腔注射MPTP（30mg/kg）；②模型组（Medi+MPTP）小鼠每日同一时间点腹腔注射介质(与雷帕霉素等体积)*7d，第3-7日，介质注射后30min时腹腔注射MPTP（30mg/kg）；③预处理对照组（Rap+Sali）小鼠每日同一时间点腹腔注射雷帕霉素(7.5mg/kg)*7d，第3-7日，雷帕霉素注射后30min时腹腔注射生理盐水（与MPTP等体积）；④空白对照组(Medi+Sali)小鼠每日同一时间点注射介质(7.5mg/kg)*7d，第3-7日，介质注射后30min时腹腔注射生理盐水。注射完毕后，在每日同一时间点观察各组小鼠行为学表现，最后一次注射MPTP或生理盐水1h后取脑组织；运用免疫荧光观察小鼠中脑黑质和纹状体P-4EBP1和a-synuclein表达；采用Western blote定性定量检测分析小鼠中脑黑质和纹状体P-4EBP1和a-synuclein变化及关系。分析探讨：①1-甲基-4-苯基-1,2,3,6-四氢吡啶（1-methyl-4-phenyl-1,2,3,6 tetrahydrop-yridine，MPTP）诱导的亚急性PD小鼠模型中，P-4EBP1蛋白和a-synuclein在中脑黑质和纹状体处的表达和关系；②TOR信号通路抑制剂雷帕霉素对MPTP小鼠亚急性PD模型P-4EBP1蛋白和a-synuclein表达的影响。结果：（1）行为学观察结果：预处理模型组较模型组行为学表现明显减轻；空白对照组和预处理对照组未见PD行为学表现。（2）免疫荧光染色结果：在预处理模型组和模型组P-4EBP1及α-synuclein表达较空白对照组和预处理对照组增加；预处理模型组P-4EBP1和α-synuclein表达较模型组降低。（3）Western blote检测蛋白结果：在预处理模型组和模型组P-4EBP1及α-synuclein表达较空白对照组和预处理组增加，，差异有统计学意义（P0.01）；预处理模型组P-4EBP1和α-synuclein表达较模型组降低，差异有统计学意义（P0.01）。结论：（1）实验结果显示：在亚急性MPTP小鼠PD模型中，TOR信号通路通过P-4EBP1参与PD发病。（2）实验结果表明：TOR信号抑制剂可能通过负调节P-4EBP1表达，降低在亚急性MPTP小鼠PD模型中α-synuclein表达，改善其行为学表现。（3）P-4EBP1可能是临床治疗PD的潜在靶点，雷帕霉素可能对临床PD有潜质保护作用。
[Abstract]:Aim: (1) to investigate the relationship between P-4EBP1 and a-synuclein expression in subacute MPTP mice. (2) to investigate whether rapamycin, a TOR inhibitor, affects a-synuclein expression in subacute MPTP mice through P-4EBP1 and TOR signaling pathways. Methods: a subacute MPTP Parkinson's disease (Parkinson's Disease,PD) mouse model (C57/BL) was used in this study. 48 mice were randomly divided into two groups: (1) pre-treatment model group (Rapamycin and MPTP,Rap MPTP),-2 model group (Medium and MPTP, Medi MPTP), 3 pretreatment control group (Rapamycin andSaline,Rap Sali), 4 blank control group (Medium andSaline, Medi Sali).) The mice in the model group were injected with rapamycin (7.5mg/kg) at the same time point every day for 7 days, and MPTP (30mg/kg) was injected intraperitoneally at the time of 30min injection on the 3rd-7th day. (2) (Medi MPTP) mice in the model group received intraperitoneal injection of MPTP (30mg/kg) at the same time point every day for 7 days, and MPTP (30mg/kg) was injected intraperitoneally on the 3rd-7th day after 30min. (3) (Rap Sali) mice in pretreatment control group were intraperitoneally injected with rapamycin (7.5mg/kg) for 7 days at the same time every day. On the 3rd-7th day, normal saline (equal to MPTP) was injected intraperitoneally at 30min after rapamycin injection. (4) (Medi Sali) mice in blank control group received intraperitoneal injection of saline at the same time point every day for 7 days, from 3 to 7 days. After 30min injection, normal saline was injected intraperitoneally. After the injection, the behavior of the mice in each group was observed at the same time every day. The brain tissue was taken after the last injection of MPTP or saline for 1 hour, and the expression of P-4EBP1 and a-synuclein in the substantia nigra and striatum of the mice was observed by immunofluorescence. The changes of P-4EBP1 and a-synuclein in substantia nigra and striatum of mice were analyzed by Western blote. Analysis and study: in subacute PD mice model induced by 11-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (1-methyl-4-phenyl-1, 2, 3, 6 tetrahydrop-yridine,MPTP), the results showed that 11-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine induced subacute PD mice. Expression and relationship of P-4EBP1 protein and a-synuclein in substantia nigra and striatum of midbrain; Effects of rapamycin, an inhibitor of 2TOR signaling pathway, on the expression of P-4EBP1 protein and a-synuclein in subacute PD model of MPTP mice. Results: (1) Behavioral observation results: the behavioral performance of preconditioned model group was significantly lower than that of model group, but no behavioral manifestation of PD was found in blank control group and pretreatment control group. (2) the results of immunofluorescence staining: the expression of P-4EBP1 and 伪-synuclein in the pretreatment model group and model group was higher than that in the blank control group and the pretreatment control group, and the expression of P-4EBP1 and 伪-synuclein in the pretreatment model group was lower than that in the model group. (3) the expression of P-4EBP1 and 伪-synuclein in the pretreatment model group and the model group were significantly higher than those in the blank control group and the pretreatment group (P 0.01); The expression of P-4EBP1 and 伪-synuclein in the pretreatment model group was lower than that in the model group, and the difference was statistically significant (P0.01). Conclusion: (1) the results showed that TOR signaling pathway was involved in the pathogenesis of PD through P-4EBP1 in subacute MPTP mice PD model. (2) the results showed that TOR signal inhibitor could negatively regulate the expression of P-4EBP1, decrease the expression of 伪-synuclein and improve its behavior in subacute MPTP mice PD model. (3) P-4EBP1 may be a potential target for clinical treatment of PD. Rapamycin may have potential protective effect on clinical PD.
【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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