【作者】 王丽；

【导师】 刘昕；

【作者基本信息】 兰州大学 ， 基础医学·病理学与病理生理学， 2014， 硕士

【摘要】 目的:复制阿尔茨海默病(AD)动物模型,观察藏药柳茶提取物对动物大脑皮层及海马区神经元损伤性病变的治疗效应,并比较经药物干预后相关蛋白质的差异性表达,从蛋白质组学的角度对柳茶治疗的作用机制进行初步探讨。方法:依随机数字表法将SD大鼠随机分为2组,A组为AD模型组、B组为柳茶提取物治疗组。肉眼观察两组大鼠一般指标:神态意识、皮毛触感、进食状况及活动能力等;制作海马和皮层区病理切片,光镜下观察海马及皮层组织神经原纤维缠结、淀粉样蛋白沉积及神经元丢失等损伤状况;以蛋白质组学技术比较AD模型组和柳茶提取物治疗组海马区蛋白质的差异性表达。结果:A组动物精神萎靡,倦怠,食欲欠佳,皮毛粗糙;B组动物灵活好动,食欲增加,皮毛较顺滑。A组大鼠海马及皮层组织可见核深染、固缩,神经元丢失,并伴有神经纤维缠结及淀粉样蛋白沉积等典型AD病理形态学变化。B组海马皮层组织神经元排列较整齐、紧密,少见沉积的淀粉样蛋白和神经纤维缠结。应用蛋白质组学技术检测到,与模型组相比柳茶提取物治疗组海马区高表达的蛋白质有8个,低表达的蛋白有1个。结合生物信息学进行质谱鉴定,确定高表达的8个蛋白质分别是突触融合蛋白-1(Complexin-1);突触融合蛋白-2(Complexin-2);髓鞘碱性蛋白(myelin basic protein,MBP);α-烯醇化酶(Enolase ENOA);苹果酸脱氢酶(Malate dehydrogenase,MDHM);琥珀酸脱氢酶[辅酶]铁硫亚基(succinate dehydrogenase complex,subunit B,iron sulfur SDHB);突触蛋白Ⅰ(SynapsinⅠ);冬氨酸氨基转移酶(Aspartate Transaminase,AATM)。而低表达的蛋白质是角蛋白Ⅱ型细胞骨架6A(Keratin,type Ⅱ cytoskeletal 6A,K2C6A)。上述差异表达蛋白质中突触融合蛋白-1、突触融合蛋白-2、髓鞘碱性蛋白和突触蛋白Ⅰ为神经系统相关蛋白;α-烯醇化酶、苹果酸脱氢酶、琥珀酸脱氢酶[辅酶]铁硫亚基和天冬氨酸氨基转移酶为代谢相关蛋白:而角蛋白Ⅱ型细胞骨架6A则属细胞骨架蛋白。结论:柳茶提取物可有效减轻阿尔茨海默病大鼠大脑皮层和海马区的三大病理变化,即神经元丢失、淀粉样蛋白沉积和神经纤维缠结的病变程度,使动物一般状况得到改善。蛋白质组学研究结果显示,经柳茶干预后有多种参与神经元以及神经系统组成的相关蛋白质、糖酵解相关的代谢酶类和细胞骨架蛋白的表达发生了改变,提示该药可能具有多靶点治疗作用,这可能是柳茶治疗阿尔茨海默病的作用机制之一。更多还原

【Abstract】 Objective:To replicate an animal model of Alzheimer’s disease(AD)to observe the therapeutic effect of Tibetan liu tea extract on cortical and hippocampal neuronal injury,and to discuss the mechanism of action of Tibetan liu tea extract in treatment from the perspective of proteomics by comparing the differential expression of related proteins after drug intervention.Methods:The SD rats were randomly divided into 2 groups:Group A(AD model group)and Group B(treatment with Tibetan liu tea extract),according to the random number table.The general states of rats in the 2 groups,such as mental state,fur,feeding behaviour and activities,were observed;Then the pathological sections were examined under optical microscope to observe cortical and hippocampal neurofibrillary tangles,amyloid depositions,neuronal loss and other damages;Finally,the differential expression of proteins in hippocampus of rats was compared between the model group and the treatment group with the technology of proteomics.Results:The rats in Group A exhibited listlessness,fatigue,poor appetite,and rough fur;In contrast,the rats in Group B exhibited restless,flexible behaviour,increased appetite,and smooth fur.Nuclear hyperchromatism,pyknosis,and neuronal loss were observed in the hippocampus and cortex of rats in Group A;Neurofibrillary tangles,amyloid protein deposition and other typical pathomorphological changes in animal brains with AD were also apparent in Group A.On the contrary,the neurons were arranged regularly and densely in the hippocampus and cortex of rats in Group B;Neurofibrillary tangles and amyloid protein deposition were infrequent in Group B.Compared with the model group(Group A),there were eight proteins with high expression and one protein with low expression in the hippocampus of rats in the treatment group,which was detected by the technology of proteomics.According to the mass spectrometry analysis with bioinformatic methods,it can be confirmed that the eight proteins with high expression are respectively Complexin-1,Complexin-2,myelin basic protein(MBP),α-enolase(ENOA),malate dehydrogenase(MDHM).succinate,dehydrogenase complex,subunit B,iron sulfur(SDHB),Synapsin I,Aspartate transaminase;and the one protein with low expression is Keratin,type Ⅱ cytoskeletal 6A(K2C6A).In all of these proteins,Complexin-1,Complexin-2,MBP and Synapsin I were nervous system relative proteins;ENOA,MDHM,SDHB and AATM were metabolism relative proteins;K2C6A was cytoskeletal protein.Conclusion:Tibetan liu tea extract can effectively relieve the symptoms such as neuronal loss,amyloid protein deposition,and neurofibrillary tangles in the hippocampus and cortex of rats with Alzheimer’s disease,and improve the condition of these rats.The results of proteomics research revealed that the expression of multiple proteins,including nervous system relative proteins,glycolytic enzymes,and cytoskeletal protein,changed after Tibetan willow tea extract intervention,which suggested that this drug has a multi-target therapeutic effect and it might be one of the mechanisms in the treatment of Alzheimer’s disease.更多还原