【摘要】：目的： （1）探討亞急性MPTP模型小鼠中P-4EBP1及與a-synuclein表達之間的關系。 （2）探討TOR抑制劑雷帕霉素是否通過P-4EBP1及TOR信號通路影響亞急性MPTP模型小鼠a-synuclein表達。 方法：本研究采用亞急性MPTP帕金森病（Parkinson's Disease，PD）小鼠（C57/BL）模型，將48只小鼠按完全隨機分組的方法分為：①預處理模型組（Rapamycin and MPTP，Rap+MPTP）、②模型組（Medium and MPTP，Medi+MPTP）、③預處理對照組（Rapamycin andSaline，Rap+Sali）、④空白對照組(Medium and Saline，Medi+Sali)。在同等條件下飼養(yǎng)：①預處理模型組小鼠每日同一時間點注射雷帕霉素(7.5mg/kg)*7d，第3-7日，介質注射后30min時腹腔注射MPTP（30mg/kg）；②模型組（Medi+MPTP）小鼠每日同一時間點腹腔注射介質(與雷帕霉素等體積)*7d，第3-7日，介質注射后30min時腹腔注射MPTP（30mg/kg）；③預處理對照組（Rap+Sali）小鼠每日同一時間點腹腔注射雷帕霉素(7.5mg/kg)*7d，第3-7日，雷帕霉素注射后30min時腹腔注射生理鹽水（與MPTP等體積）；④空白對照組(Medi+Sali)小鼠每日同一時間點注射介質(7.5mg/kg)*7d，第3-7日，介質注射后30min時腹腔注射生理鹽水。注射完畢后，在每日同一時間點觀察各組小鼠行為學表現，最后一次注射MPTP或生理鹽水1h后取腦組織；運用免疫熒光觀察小鼠中腦黑質和紋狀體P-4EBP1和a-synuclein表達；采用Western blote定性定量檢測分析小鼠中腦黑質和紋狀體P-4EBP1和a-synuclein變化及關系。分析探討：①1-甲基-4-苯基-1,2,3,6-四氫吡啶（1-methyl-4-phenyl-1,2,3,6 tetrahydrop-yridine，MPTP）誘導的亞急性PD小鼠模型中，P-4EBP1蛋白和a-synuclein在中腦黑質和紋狀體處的表達和關系；②TOR信號通路抑制劑雷帕霉素對MPTP小鼠亞急性PD模型P-4EBP1蛋白和a-synuclein表達的影響。 結果： （1）行為學觀察結果：預處理模型組較模型組行為學表現明顯減輕；空白對照組和預處理對照組未見PD行為學表現。 （2）免疫熒光染色結果：在預處理模型組和模型組P-4EBP1及α-synuclein表達較空白對照組和預處理對照組增加；預處理模型組P-4EBP1和α-synuclein表達較模型組降低。 （3）Western blote檢測蛋白結果：在預處理模型組和模型組P-4EBP1及α-synuclein表達較空白對照組和預處理組增加，，差異有統(tǒng)計學意義（P0.01）；預處理模型組P-4EBP1和α-synuclein表達較模型組降低，差異有統(tǒng)計學意義（P0.01）。 結論： （1）實驗結果顯示：在亞急性MPTP小鼠PD模型中，TOR信號通路通過P-4EBP1參與PD發(fā)病。 （2）實驗結果表明：TOR信號抑制劑可能通過負調節(jié)P-4EBP1表達，降低在亞急性MPTP小鼠PD模型中α-synuclein表達，改善其行為學表現。 （3）P-4EBP1可能是臨床治療PD的潛在靶點，雷帕霉素可能對臨床PD有潛質保護作用。
[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 偽-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 偽-synuclein in the pretreatment model group was lower than that in the model group. (3) the expression of P-4EBP1 and 偽-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 偽-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 偽-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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R742.5

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