本文關(guān)鍵詞： 帕金森病(PD) 電針 自噬 酪氨酸羥化酶(TH) α-突觸核蛋白(α-syn)　出處：《湖北中醫(yī)藥大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:采用頸背部皮下注射魚藤酮法制備帕金森病(Parkinson’s Disease,PD)模型,觀察電針治療對PD模型大鼠自噬相關(guān)蛋白微管相關(guān)蛋白1輕鏈3Ⅱ(Microtubule-associated protein 1 light chain3,LC3-Ⅱ)、P62表達的影響,探討自噬在針刺防治PD中的作用。方法:選用SPF級健康雄性SD大鼠120只,體重(180±20)g,適應(yīng)性喂養(yǎng)1周后可進行分組。8組分別是正常組,模型組,電針治療7d組,假手術(shù)組,電針治療14d組,電針治療21d組,電針治療28d組,電針預治療組,每組15只。采用頸背部皮下注射魚藤酮法制備PD模型,給予魚藤酮(2mg/kg,溶解于葵花油,濃度2mg/ml,充分混勻并避光保存)頸背部皮下注射,1次/d,連續(xù)28d。正常組不做任何處理;假手術(shù)組方法同模型組,但只注射不含有魚藤酮的等量葵花油乳化劑(溶劑);電針預治療組首先對正常的大鼠進行電針治療7d,其后造模。4個電針治療組模型建立結(jié)束之后,根據(jù)穴位太沖以及風府,進行電針治療。選擇使用0.5寸毫針進行針刺,接通G6805電針治療儀,連續(xù)波,頻率設(shè)為120次/分鐘,每次治療20min,每天1次,療程分別為7d、14d、21d、28d。其中“風府”為每次固定使用,“太沖”隔天左右交替使用。(1)對各組大鼠進行行為學評分、運用敞箱實驗檢測各組大鼠水平運動以及垂直運動情況。(2)運用免疫組織化學染色法檢測各組大鼠中腦黑質(zhì)區(qū)酪氨酸羥化酶(Tyrosine hydroxylase,TH)、α-突觸核蛋白(α-synuclein,α-syn)陽性表達。(3)運用RT-PCR法檢測各組大鼠中腦黑質(zhì)區(qū)LC3-Ⅱm RNA、P62m RNA表達的變化。(4)運用Western blot法檢測各組大鼠中腦黑質(zhì)區(qū)LC3-Ⅱ、P62蛋白表達的變化。結(jié)果:(1)評分結(jié)果示:與正常組及假手術(shù)組相比,模型組大鼠行為學評分明顯升高(P0.01);與模型組相比,各電針治療組及電針預治療組大鼠行為學評分顯著降低(均P0.01)。敞箱實驗結(jié)果示:模型組的大鼠水平運動、垂直運動活性跟正常組以及假手術(shù)組比明顯下降(P0.01);各電針治療組及電針預治療組PD模型大鼠敞箱實驗水平運動和垂直運動活性較模型組改善明顯(均P0.01)。(2)免疫組化檢測結(jié)果示:與正常組及假手術(shù)組相比,模型組大鼠黑質(zhì)內(nèi)TH平均光密度值明顯降低,α-syn平均光密度值明顯增高(均P0.01);與模型組相比,各電針治療組及電針預治療組大鼠黑質(zhì)內(nèi)TH平均光密度值顯著升高,α-syn平均光密度值顯著降低(均P0.01);正常組及假手術(shù)組大鼠黑質(zhì)內(nèi)TH和α-syn平均光密度值無明顯差異(P0.05)。(3)RT-PCR檢測結(jié)果示:與正常組及假手術(shù)組相比,模型組大鼠黑質(zhì)內(nèi)LC3-IIm RNA、P62m RNA表達顯著升高(均P0.01);與模型組相比,電針治療28d組、電針治療21d組、電針治療14d組LC3-IIm RNA表達顯著升高,P62m RNA表達則顯著降低(均P0.01),電針預治療組和電針治療7d組大鼠黑質(zhì)內(nèi)LC3-IIm RNA、P62m RNA表達與模型組相比未見明顯差異(均P0.05)。(4)Western blot檢測結(jié)果示:與正常組及假手術(shù)組相比,模型組LC3-Ⅱ、P62蛋白表達均明顯增加(均P0.01)。與模型組相比,各電針治療組及電針預治療組LC3-Ⅱ蛋白表達水平增加明顯,P62蛋白表達有顯著下調(diào)(均P0.01)。結(jié)論:自噬的激活及功能的正常運作能有效清除細胞內(nèi)沉積的α-syn,延緩PD的進程。電針治療可以改善自噬障礙,促使自噬流的正常運行,促進具有細胞毒性作用α-syn的降解,從而可對DA能神經(jīng)元發(fā)揮著保護作用。
[Abstract]:Objective: the preparation of Parkinson's disease by subcutaneous injection of rotenone (Parkinson 's Disease, by PD) model to study the effect of Electroacupuncture on autophagy related protein PD in the rat model of microtubule associated protein 1 light chain 3 (Microtubule-associated protein 1 light chain3 II, LC3- II), expression of P62, to investigate the role of autophagy in the acupuncture treatment of PD in the selection of grade SPF. Methods: 120 healthy male SD rats, weight (180 + 20) g, after 1 weeks of feeding can be grouped in group.8 were normal group, model group, electroacupuncture treatment 7d group, sham operation group, electroacupuncture treatment 14d group, electroacupuncture treatment 21d group, electroacupuncture treatment 28d group, electroacupuncture treatment group, 15 rats in each group. The model of PD by using subcutaneous injection of rotenone was given rotenone (2mg/kg, dissolved in sunflower oil, the concentration of 2mg/ml, mixing and save) neck back subcutaneous, 1 /d, 28d. normal group did not receive any. Daniel; sham operation group with the model group, but only the equivalent of sunflower oil emulsifier injection does not contain rotenone (solvent); electroacupuncture treatment group were treated with electro acupuncture on normal rats 7d first, then after modeling.4 EA group model established the end of treatment, according to the acupoint and Fengfu Taichong, electro acupuncture treatment. Choose to use 0.5 inch haozhen acupuncture, electro acupuncture on G6805, continuous wave, frequency is 120 times / min, each treatment 20min, 1 times a day, treatment were 7d, 14d, 21d, 28d. and "Fengfu" for each fixed use, "Taichong" about the alternate use of (1). Scores of rats in each group were using the open field behavior, experimental rats were detected and the vertical movement of horizontal movement. (2) by using immunohistochemical staining method to detect tyrosine hydroxylase in rat midbrain substantia nigra (Tyrosine hydroxylase, TH), alpha synuclein White (alpha -synuclein, alpha -syn) positive expression. (3) using RT-PCR method to detect the rat midbrain substantia nigra of LC3- II m RNA, changes of P62m RNA expression. (4) using Western blot method to detect the rat substantia nigra in LC3- II, changes in the expression of P62 protein. Results: (1) score results show: compared with normal group and sham operation group, model group rats behavioral score increased significantly (P0.01); compared with the model group, rats of group behavior score was significantly lower in the electroacupuncture treatment group and electroacupuncture treatment (P0.01). The open field experiment results showed: the model group rats horizontal movement, vertical movement activity with normal group and sham operation group decreased significantly (P0.01); the electro acupuncture group and electroacupuncture treatment group PD rat model of open field test of horizontal movement and vertical movement activity was obviously improved compared with the model group (P0.01). (2) the immunohistochemical results showed: compared with normal group and sham operation group, The model group of rat substantia nigra in the average optical density of TH was decreased and a -syn average optical density value was significantly increased (P0.01); compared with the model group, the electroacupuncture treatment group and electroacupuncture treatment group rat substantia nigra in the average optical density of TH was significantly increased, a -syn average optical density decreased significantly (P0.01); normal group and sham operation group rats in substantia nigra and TH alpha -syn average optical density had no significant difference (P0.05). (3) the results of RT-PCR showed that: compared with normal group and sham operation group, model group of rat substantia nigra in LC3-IIm RNA, P62m RNA expression was significantly increased (all P0.01); compared with the model group, electroacupuncture treatment 28d group, electroacupuncture treatment 21d group, electroacupuncture treatment group 14d LC3-IIm significantly increased RNA expression, P62m RNA expression decreased significantly (P0.01), electroacupuncture treatment group and electroacupuncture group 7d rat substantia nigra in LC3-IIm RNA, P62m RNA expression compared with the model group there was no significant difference (all P0.05 (4) Western). The results of blot showed that: compared with normal group and sham operation group, model group, LC3- II, P62 protein expression increased significantly (P0.01). Compared with the model group, the electroacupuncture treatment group and electroacupuncture treatment group II LC3- protein expression level increased significantly, the expression of P62 protein significantly cut (P0.01). Conclusion: a normal operation of -syn activation and function of autophagy can effectively remove intracellular deposition, delay the process of PD. Acupuncture therapy can improve the normal operation of the autophagy autophagy disorder, flow, promote the cytotoxic effect of alpha -syn degradation, which can play a protective effect on DA neurons.

【學位授予單位】：湖北中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R245;R-332

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