本文選題：帕金森 + MPTP�。� 參考：《南京中醫(yī)藥大學》2017年碩士論文

【摘要】：目的:通過1-甲基-4-苯基-1,2,3,6-四氫吡啶(1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride,MPTP)慢 性帕金 森小鼠 模型,觀察松果菊苷(Echinacoside,ECH)能否通過自噬途徑緩解模型小鼠多巴胺(Dopamine,DA)神經(jīng)元的損傷,并探討松果菊苷通過自噬途徑保護DA能神經(jīng)元的作用機制及松果菊苷發(fā)揮自噬作用可能的信號通路,為溫腎養(yǎng)肝方在帕金森病的臨床運用提供理論和科學依據(jù)。方法:選用10周齡C57BL/6J雄性小鼠,體重為25～30g,固定時間給予松果菊苷(Echinacoside,ECH)(30mg/kg)/司來吉蘭(Selegiline,SEL)(1mg/kg)灌胃處理,1h后給予皮下注射MPTP0.1ml/10g,每周兩次,持續(xù)四周,建立MPTP慢性帕金森小鼠模型。通過行為學觀察(爬桿實驗、步態(tài)分析)評價小鼠運動遲緩和平衡協(xié)調(diào)能力及步態(tài)異常。運用酪氨酸輕化酶(tyrosine hydroxylase,TH)免疫組化方法,并結(jié)合體視學計數(shù),分析模型小鼠黑質(zhì)部(Substantia nigra,SN)DA能神經(jīng)元的損傷狀況。應用蛋白免疫印跡法,檢測自噬相關蛋白mTOR(Mammalian target of rapamycin)的蛋白表達水平,Beclin-1的蛋白表達水平,P62的蛋白表達水平,LC3-Ⅱ(Chain3-Ⅱ)的蛋白表達水平,α-突觸核蛋白(α-synuclein,a-SN)的蛋白表達水平,及PI3K(PhosphatidylInositol3-kinase)、AKT(Protein kinase B)、Atg13(autophagy-related gene 13)磷酸化的蛋白表達水平。結(jié)果:行為學分析提示松果菊苷能夠減少模型小鼠爬桿總時間(桿上停留時間及爬桿時間,P0.05),但步態(tài)分析未提供統(tǒng)計學差異(P0.05)。免疫組化提示松果菊苷能減少MPTP慢性帕金森小鼠DA的損傷(P0.01)。蛋白免疫印跡法提示松果菊苷能逆轉(zhuǎn)MPTP慢性模型小鼠中腦Beclin-1表達下調(diào)(P0.05),下調(diào)MPTP慢性模型小鼠中腦P62的表達(P0.05),促進MPTP慢性模型小鼠中腦LC3-Ⅱ表達增加(P0.05),下調(diào)MPTP慢性模型小鼠中腦α-synuclein的表達(P0.05),但PI3K、AKT、mTOR、Atg13磷酸化的蛋白表達水平未提供統(tǒng)計學差異(P0.05)。結(jié)論:松果菊苷能夠通過自噬途徑發(fā)揮對MPTP誘導的慢性PD模型小鼠中腦DA神經(jīng)元的保護作用。
[Abstract]:Objective: to observe whether Echinacoside ECH can attenuate the injury of dopamine DA neurons in mouse model by using 1-Methyl-4-phenyl-6-tetrahydropyridine hydrochloridede MPTP (1-Methyl-4-phenyl-1-tetrahydropyridine hydrochloridede-MPTP) model in Parkinsen mice model by means of 1-methyl-4-phenyl-6-tetrahydropyridine hydrochloridedeform-MPTP (1-Methyl-4-phenyl-1-tetrahydropyridine) -1-methyl-4-phenyl-6-tetrahydropyridine hydrochloridedede-MPTP (MPTP) model. To explore the mechanism of pyrethrin protecting DA neurons through autophagy and the possible signal pathway of autophagy, which provides a theoretical and scientific basis for the clinical application of Wenshen Yanggan recipe in Parkinson's disease. Methods: 10-week-old C57BL / 6J male mice, weighing 25g / 30g, were treated with 30mg/kg / 1mg/kg for 1 h, then subcutaneously injected MPTP 0.1ml / 10g, twice a week for four weeks, to establish the model of chronic Parkinson's disease in mice. The behavioral observation (pole climbing test, gait analysis) was used to evaluate the motor retardation, balance and coordination ability and gait abnormality in mice. The damage of DA neurons in substantia nigra SN of model mice was analyzed by using tyrosine hydroxylase th immunohistochemical method and stereological counting. Western blot was used to detect the protein expression level of autophagy associated protein mTOR and Beclin-1 protein. The protein expression level of P62 and 偽 -synucleinina-SN were determined by Western blotting, and the protein expression of LC3- 鈪，

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