【作者】 王欲立；

【导师】 聂莹雪；

【作者基本信息】 中国医科大学 ， 神经病学， 2010， 硕士

【摘要】 目的帕金森病(PD)是一种常见的与年龄相关的神经变性疾病,主要特征是黑质多巴胺能神经元的进行性丢失和变性。JNK信号转导通路是MAPK通路的一条重要分支,它在细胞周期、生产、凋亡和细胞应激等多种生理和病理过程中起重要作用。目前研究证据表明帕金森病发生有JNK信号转导通路的参与。丁苯酞是从我国南方的一种水芹菜籽中分离出的有效成分,简称NBP,又名芹菜甲素,是中国医学科学院中国协和医科大学药物研究所人工合成的消旋体,从80年代开始冯亦璞带领该所开始研究NBP的药理作用：能改善缺血区局部脑血流,增加缺血区毛细血管的数量,增加缺血区脑血流量,改善缺血性脑能量代谢,缩小梗死面积,减轻神经功能损伤程度,减轻脑水肿；还可改善线粒体功能,增强呼吸链功能,能改善线粒体能量泵,增加抗氧化作用,从而发挥抗凋亡；能增加大鼠全脑缺血纹状体细胞外液氨基酸和多巴胺含量。故本试验依据JNK在PD发病的作用来研究NBP对MPTP诱导的多巴胺能神经毒性是否具保护作用。我们选择丁苯酞用于MPTP制备C57BL／6帕金森病小鼠模型,用免疫组织化学染色方法和westernblot检测帕金森病小鼠黑质多巴胺能神经元TH、DAT、p-JNK/JNK表达情况,观察并分析其表达意义。方法1、动物分组：30只C57BL／6小鼠采用完全随机分组法分为以下3组,每组10只。(1)正常对照组(NS+植物油)：腹腔注射与MPTP组等体积生理盐水5 d,再注射与治疗组等体积植物油14d。(2)MPTP组(MPTP+植物油)：采用MPTP(30 mg/kg)连续5 d腹腔注射,然后改用与治疗组等体积植物油注射14 d。(3)NBP治疗组(MPTP+NBP):先以MPTP(30 mg/kg)连续腹腔注射5 d,然后丁基苯酞(NBP40 mg/kg)腹腔注射14 d。每组动物于第19天处死后取脑组织进行固定石蜡包埋,取中脑黑质部位切片进行免疫组织化学染色；另外一侧脑组织提取出黑质后迅速置于-80℃冰箱冷冻用于westernblot。2、模型制备：MPTP(30 mg/kg):连续5 d腹腔注射,注射药物后小鼠即刻出现步态跚,易激惹,毛及鼠尾竖起等反应,约5～10 min后复常态,即视为模型制备成功。3、免疫组织化学染色方法检测TH、DAT的表达4、Westernblot方法检测p-JNK、JNK的表达结果1、与正常对照组相比,MPTP组小鼠黑质多巴胺能神经元中的TH阳性神经元表达数目明显减少(P<0.05)；与MPTP组相比,NBP治疗组小鼠黑质多巴胺能神经元中TH阳性神经元表达数目增加(P<0.05),二者均具有统计学意义。2、与正常对照组相比,MPTP组小鼠黑质纹状体多巴胺能神经纤维中的DAT阳性神经纤维表达数目明显减少(P<0.05)；与MPTP组相比,NBP治疗组小鼠黑质纹状体中DAT阳性神经纤维数目表达增加(P<0.05),二者均具有统计学意义。3、正常对照组几乎未见p-JNK表达,MPTP组可见p-JNK/JNK的表达明显增加,而且表达较正常对照组强,二者差别有统计学意义(P<0.05),NBP治疗组p-JNK/JNK表达较MPTP组下降,二者差别有统计学意义(P<0.05)。结论丁苯酞可增加帕金森病小鼠黑质多巴胺能神经元TH、DAT的表达,减少p-JNK表达,拮抗JNK信号转导通路,改善多巴胺能神经元功能,这为丁苯酞可能成为治疗PD药物的临床可能性提供了实验依据。更多还原

【Abstract】 ObjectivesParkinson’s disease (PD) is a common age-related neurodegenerative disease, mainly characterized by dopaminergic neurons loss and degeneration of the sexual. JNK signal transduction pathway is an important branch of MAPK pathway, which in the cell cycle, production, apoptosis and cell stress and other physiological and pathological processes play an important role. Current research evidence suggests that occurrence of Parkinson’s disease JNK signal transduction pathway involved. Butylphthalide is a kind of cress from rapeseed in South China isolated the active ingredient, referred to as NBP, a hormone known as celery, Chinese Academy of Medical Sciences Peking Union Medical College Institute of Chinese synthetic raceme, from the 80’s Start Feng Yipu leading the research begun by the pharmacological effects of NBP:to improve regional cerebral blood flow in ischemic region, increasing the number of capillary ischemia, ischemic cerebral blood flow increased to improve the ischemic brain energy metabolism, reduce infarct size, reduce the neurological damage and cerebral edema; can improve mitochondrial function, enhance the respiratory chain function, can improve mitochondrial energy pumps to increase anti-oxidation, anti-apoptosis and thus play; to increase global cerebral ischemia in striatal cells extracellular amino acids and dopamine content. The purpose of this experiment based on the role of JNK in the pathogenesis of PD to study the NBP on the MPTP-induced dopaminergic neurotoxicity is a protective effect. We choose butylphthalide preparation for MPTP C57BL/6 mouse model of Parkinson’s disease, by immunohistochemical staining and detection of Parkinson’s disease in mice westernblot dopaminergic neurons of TH, DAT, p-JNK/JNK expression to observe and analyze its clinical significance. Methods1、Animal group 30 C57BL/6 mice were divided into groups using a randomized complete the following 3 groups of 10 each. (1) normal control group (NS +vegetable oil):intraperitoneal injection of saline and MPTP groups 5 d, then injected with equal volume of vegetable oil treatment group 14d. (2) MPTP group (MPTP+ vegetable oil):The MPTP (30 mg/kg) intraperitoneally consecutive 5 d, and then switch to the treatment group with equal volume of vegetable oil injection 14 d. (3) NBP treated group (MPTP+NBP):first to MPTP (30 mg/kg) by intraperitoneal injection 5 d, then NBP (NBP 40 mg/kg) intraperitoneally 14 d. Each animal was killed on day 19 were fixed after the brain tissues embedded in paraffin, to take part in the substantia nigra slices by immunohistochemical staining; other side of the substantia nigra brain tissue extracted quickly placed in-80℃freezer with in westernblot.2、Model Preparation MPTP (30 mg/kg) intraperitoneally 5 d row [1], mice immediately after injection of gait limp, irritability, hair and tail erect reactions, about 5-10 min after the resumption of normal, that is, Preparation of success as a model.3、Immunohistochemical staining detected TH, DAT expression 4. Westernblot to detect p-JNK, JNK expressionResults1、With the normal control group, MPTP mice dopaminergic neurons in the TH-positive neurons and decreases the number (P<0.05); with MPTP compared, NBP treated mice substantia nigra dopaminergic neurons of TH-positive neurons increase (P <0.05), both were statistically significant.2、With the normal control group, MPTP mice nigrostriatal dopaminergic nerve fibers in the DAT positive nerve fibers were the number decreased (P<0.05); with MPTP compared, NBP treated mice nigra profile like the body of DAT-positive nerve fibers in the number of expression (P<0.05), both were statistically significant.3、The control group almost has no p-JNK expression, MPTP group showed p-JNK/JNK expression was significantly increased and expression than the normal control group strength, the two differences was statistically significant (P<0.05), NBP treated group p-JNK/JNK expression compared with MPTP group decreased, the two differences was statistically significant (P<0.05).ConclusionsButylphthalide increase Parkinsonian Mice nigral dopaminergic neurons TH, DAT expression reduce p-JNK expression, antagonistic JNK signal transduction pathway improve dopaminergic neuronal function, This butylphthalide may become treatment PD drugs clinical possibility provides experimental basis.更多还原