电针对帕金森病模型大鼠黑质内自噬相关蛋白LC3-Ⅱ、P62表达的影响研究

发布时间：2018-02-07 11:50

本文关键词： 帕金森病(PD) 电针自噬酪氨酸羟化酶(TH) α-突触核蛋白(α-syn)　出处：《湖北中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:采用颈背部皮下注射鱼藤酮法制备帕金森病(Parkinson’s Disease,PD)模型,观察电针治疗对PD模型大鼠自噬相关蛋白微管相关蛋白1轻链3Ⅱ(Microtubule-associated protein 1 light chain3,LC3-Ⅱ)、P62表达的影响,探讨自噬在针刺防治PD中的作用。方法:选用SPF级健康雄性SD大鼠120只,体重(180±20)g,适应性喂养1周后可进行分组。8组分别是正常组,模型组,电针治疗7d组,假手术组,电针治疗14d组,电针治疗21d组,电针治疗28d组,电针预治疗组,每组15只。采用颈背部皮下注射鱼藤酮法制备PD模型,给予鱼藤酮(2mg/kg,溶解于葵花油,浓度2mg/ml,充分混匀并避光保存)颈背部皮下注射,1次/d,连续28d。正常组不做任何处理;假手术组方法同模型组,但只注射不含有鱼藤酮的等量葵花油乳化剂(溶剂);电针预治疗组首先对正常的大鼠进行电针治疗7d,其后造模。4个电针治疗组模型建立结束之后,根据穴位太冲以及风府,进行电针治疗。选择使用0.5寸毫针进行针刺,接通G6805电针治疗仪,连续波,频率设为120次/分钟,每次治疗20min,每天1次,疗程分别为7d、14d、21d、28d。其中“风府”为每次固定使用,“太冲”隔天左右交替使用。(1)对各组大鼠进行行为学评分、运用敞箱实验检测各组大鼠水平运动以及垂直运动情况。(2)运用免疫组织化学染色法检测各组大鼠中脑黑质区酪氨酸羟化酶(Tyrosine hydroxylase,TH)、α-突触核蛋白(α-synuclein,α-syn)阳性表达。(3)运用RT-PCR法检测各组大鼠中脑黑质区LC3-Ⅱm RNA、P62m RNA表达的变化。(4)运用Western blot法检测各组大鼠中脑黑质区LC3-Ⅱ、P62蛋白表达的变化。结果:(1)评分结果示:与正常组及假手术组相比,模型组大鼠行为学评分明显升高(P0.01);与模型组相比,各电针治疗组及电针预治疗组大鼠行为学评分显著降低(均P0.01)。敞箱实验结果示:模型组的大鼠水平运动、垂直运动活性跟正常组以及假手术组比明显下降(P0.01);各电针治疗组及电针预治疗组PD模型大鼠敞箱实验水平运动和垂直运动活性较模型组改善明显(均P0.01)。(2)免疫组化检测结果示:与正常组及假手术组相比,模型组大鼠黑质内TH平均光密度值明显降低,α-syn平均光密度值明显增高(均P0.01);与模型组相比,各电针治疗组及电针预治疗组大鼠黑质内TH平均光密度值显著升高,α-syn平均光密度值显著降低(均P0.01);正常组及假手术组大鼠黑质内TH和α-syn平均光密度值无明显差异(P0.05)。(3)RT-PCR检测结果示:与正常组及假手术组相比,模型组大鼠黑质内LC3-IIm RNA、P62m RNA表达显著升高(均P0.01);与模型组相比,电针治疗28d组、电针治疗21d组、电针治疗14d组LC3-IIm RNA表达显著升高,P62m RNA表达则显著降低(均P0.01),电针预治疗组和电针治疗7d组大鼠黑质内LC3-IIm RNA、P62m RNA表达与模型组相比未见明显差异(均P0.05)。(4)Western blot检测结果示:与正常组及假手术组相比,模型组LC3-Ⅱ、P62蛋白表达均明显增加(均P0.01)。与模型组相比,各电针治疗组及电针预治疗组LC3-Ⅱ蛋白表达水平增加明显,P62蛋白表达有显著下调(均P0.01)。结论:自噬的激活及功能的正常运作能有效清除细胞内沉积的α-syn,延缓PD的进程。电针治疗可以改善自噬障碍,促使自噬流的正常运行,促进具有细胞毒性作用α-syn的降解,从而可对DA能神经元发挥着保护作用。
[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.

【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245;R-332

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