【作者】 高宁；

【导师】 秦川； 张连峰；

【作者基本信息】 中国协和医科大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 帕金森病(Parkinson’s disease,PD)是一种年龄相关的慢性进行性神经系统疾病,静止性震颤、强直、运动徐缓、步态不稳等是其主要的临床特征,现在已成为威胁老年人健康的主要疾病之一。帕金森病主要病理特征表现为黑质纹状体多巴胺能神经元变性和缺失,细胞内包含体—路易小体(Lewy body,LB)的形成。α-Synuclein是帕金森病特征性病变路易小体的主要成分,在帕金森的发病机制中发挥重要作用。生理条件下,它可调节突触血管的再生和重塑,同时在脂质代谢,信号转导及囊泡转运等方面也有重要作用。研究发现早发型家族遗传性帕金森病患者出现α-synuclein基因的错义突变A53T和A30P,及野生型二倍体表达增多。突变的α-synuclein可促进自身寡聚物的形成,增加其神经毒性。但是,突变及过量表达的α-synuclein究竟是通过何种分子机制促进了PD的发生至今仍不清楚。因此建立α-synuclein转基因动物模型可为今后帕金森病的研究提供有力的模型基础。为了更好的研究α-synuclein在PD发病过程中的作用,本实验采用神经特异性启动子PDGF,建立了在神经系统特异性表达人α-synuclein野生型及两个突变型A53T和A30P的转基因小鼠。构建人α-synuclein表达载体,利用显微注射法制备人α-synuclein基因的转基因小鼠。通过PCR方法鉴定转基因首建鼠及其子代基因型。通过RT-PCR和Western blotting方法鉴定转基因小鼠脑组织中人α-synuclein mRNA和蛋白表达情况。得到表达水平不同的野生型人α-synuclein转基因小鼠2个品系。得到表达水平不同的A53T突变型α-synuclein转基因小鼠2个品系。得到表达水平不同的A30P突变型α-synuclein转基因小鼠3个品系。采用免疫组化鉴定人α-synuclein在小鼠脑组织中的表达情况。实验结果显示,转基因小鼠大脑海马、新皮层、纹状体区出现人α-synuclein阳性标记的细胞。通过Rota rod实验评价转基因小鼠的行为改变情况,发现三种转基因小鼠运动能力均有明显下降,A53T,A30P和SYN-WT三种转基因小鼠与阴性对照鼠相比分别下降45.4%,46.2%和45.5%。以上结果显示,我们已成功建立人α-synuclein转基因小鼠帕金森病模型,可为今后帕金森病发病机制的研究及帕金森病药物的开发提供模型基础。但α-synuclein对帕金森病作用机制尚有许多不明之处,还需做进一步的研究。更多还原

【Abstract】 Parkinson’s disease (PD) is a chronically progressive, age-related and fatal neurological disease. Resting tremor, rigidity, bradykinesia and postural instability characterize PD clinically. It has become one of the major diseases which threaten the health of the seniors. The pathological hall-marks of PD are degeneration and elimination of dopamine neurons in substantia nigra as well as the formation of intracellular inclusion, known as Lewy body (LB).α-Synuclein is the major constituent of Lewy bodies which play an important role in the pathogenesis of Parkinson’s disease. Under normal conditions, it is thought to have a role in the modulation of synaptic vesicle turnover and synaptic plasticity. And it is also important in lipid metabolism, signal transduction and vesicle transport. Two missense mutations in theα-synuclein gene (Ala-53→Thr and Ala-30→Pro) have been found in rare autosomal dominant inherited forms of PD. Duplication and triplication in theα-synuclein gene are also causes of PD. Mutation ofα-synuclein can promote the formation of oligomer to increase their neurotoxity. However, the mutation and excessive expression ofα-synuclein is through which molecular mechanism for the occurrence of PD still unclear. Therefore the establishment ofα-synuclein transgenic animal model of Parkinson’s disease may provide an important model for research in the future.In order to research the role ofα-synuclein in PD pathogenesis, the humanα-Synuclein wild type gene, A53T mutation and A30P mutation transgenic mice were generated. The PDGF driving vectors expressing theα-Synuclein wild type gene, A53T mutation and A30P mutation were constructed. The transgenic mice were created with the microinjection method. The genotype of transgenic founders and its filial generations were identified by PCR. The expression level of humanα-synuclein was analyzed by RT-PCR and Western blotting. Two humanα-synuclein WT transgenic lines with different expression levels were established. Two humanα-synuclein A53T transgenic lines with different expression levels were established. The three humanα-synuclein A30P transgenic lines with different expression levels were established either. The expression levels of humanα-synuclein in the brains of transgenic mice were analyzed by immunohistochemistry staining. The expression ofα-synuclein was detected in hippocampus, neocortex and substantia nigra of all the 7 lines. The decline in motor performance of transgenic mice was measured by rotating rod experiment. All of the transgenic mice lines showed obviously progressive decline in motor performance, A53T, A30P and SYN-WT transgenic mice were down 45.4%, 46.2% and 45.5% compared with the negative control mice.These results indicate that, we have successfully established humanα-synuclein transgenic mouse model of Parkinson’s disease. Although, there are still many unknown mechanisms ofα-synuclein in Parkinson’s disease, further research needs to be done, the basis model for the pathogenesis research and drug development of Parkinson’s disease could be provided in the future.更多还原