环境毒素联合VMAT2遗传操控建立帕金森病动物模型的研究

Parkinson's disease (PD), the etiology of which remains unknown, is a common neurodegenerative disorder that seriously affects the sufferer's quality of life. None of the current neurotoxin-based or genetic-based animal model of PD recapitulates aging-gene-environment interaction pattern of PD pathogenesis, which becomes a conspicuous issue that halts the progression of PD research. Model that could better mimic the etiology and pathogenesis of PD is needed urgently to overcome the bottleneck of PD research. It has been indicated that transgenic mice which remain 5% of VMAT2 protein activity present the motor-, non-motor symptoms and pathology of α-synuclein accumulation, but only at 28 months old or elder. Furthermore, in our PD clinic, we found a PD patient who has experienced a history of eating fenpropathrin-poisoned fish for half a year. Subsequently, we laboratorily confirmed the positive correlation between fenpropathrin and PD in dopaminergic cell cultures, mice and rats. In this study, we propose to develop PD models based on ageing, VMAT2 gene susceptibility and chronic fenpropathrin or rotenone exposure in dopaminergic cell lines, caenorhabditis elegans and mice. The behavior assessments, brain histology, peripheral toxicity, biogenic amine levels in the striatum, the ultrastructural changes of the SNpc, and the oxidative stress levels of the midbrain are employed to verify the reliability of these models. We aim to establish a reliable and more clinically-related model, which could overcome the limitations of current PD models and promote the progression of PD research, especially for the pathogenesis and disease-modifying treatment research of PD.

帕金森病（PD）是病因未明的常见神经变性病。现有的神经毒素和遗传模型都未能充分反映PD的年龄、遗传和环境因素交互作用的发病特征，已成为制约PD研究进步的主要障碍之一。建立能更好模拟PD发病机制的动物模型是该领域亟待突破的问题。研究表明，降低VMAT2表达至正常5%水平的基因敲低小鼠可以较全面呈现PD的运动、非运动症状和α-突触核蛋白聚集病理，但耗时长达28个月。本课题拟在细胞、线虫和小鼠中，探索敲低VMAT2表达联合我们在临床上首次发现并经过体内外研究验证的新环境毒素甲氰菊酯或经典毒素鱼藤酮为促发因素，建立一种融合年龄、以VMAT2敲低为遗传背景、甲氰菊酯或鱼藤酮慢性暴露为诱因的PD模型，通过行为学、组织病理、神经生化和分子生物等方法验证模型的可靠性，旨在弥补现有模型的不足，为进一步研究PD的发病机制和疾病修饰治疗提供快速稳定且能更好模拟人类PD发病过程的模型，推动PD研究的进步。

帕金森病（PD）是病因未明的常见神经变性病。现有的神经毒素和遗传模型都未能充分反映PD的年龄、遗传和环境因素交互作用的发病特征，已成为制约PD研究进步的主要障碍之一。建立能更好模拟PD发病机制的动物模型是该领域亟待突破的问题。本课题首先（第一部分）在SK-N-SH多巴胺能细胞系中敲低VMAT2表达并联合不同浓度的鱼藤酮处理，探索最佳化PD细胞模型。我们发现VMAT2敲低组细胞对环境毒素鱼藤酮的易感性明显增加，细胞形态皱缩，氧化应激相关蛋白OXSR1及a-synuclein表达呈增加趋势。以上结果表明VMAT2敲低细胞模型在环境毒素诱导下更易产生PD样损伤反应。随后我们（第二部分）观察并评估VMAT2不同表达水平小鼠（VMAT2 LO、VMAT2 HT、WT、VMAT2 HI）随年龄变化（2月，6月，12月，18月，30月）的非运动（抑郁样行为）及运动变化情况，及其黑质纹状体系统退化情况，实验显示VMAT2 HT小鼠6月份即出现抑郁样行为，并在18月龄及30月龄仍有差异，而VMAT2高表达水平的小鼠VMAT2 HI与WT小鼠相比未见明显差异，并且VMAT2 HT小鼠直到30月龄才表现出运动能力下降，黑质及纹状体TH神经元数目减少。随后我们运用VMAT2敲低小鼠，联合鱼藤酮及MPTP的暴露，建立稳定的PD动物模型（第三部分），我们发现针对VMAT2敲低小鼠和野生型小鼠（对照组）予鱼藤酮灌胃给药（30 mg/kg/d，持续8周）。VMAT2敲低小鼠运动及协调能力明显减退，抑郁程度增加，学习、记忆等认知功能与对照组无明显差异，而鱼藤酮干预后，VMAT2敲低小鼠认知功能则出现减退；并且黑质及纹状体DA含量显著降低，a-synuclein表达明显增加，并在胞浆内异常聚集形成路易小体样结构，12月龄老年小鼠中以上变化更为显著。同时针对六月龄的VMAT2 HT小鼠和野生型小鼠（对照组）予MPTP（30 mg/kg/d，持续7天）皮下注射（第四部分），我们发现嗅束（OB）及蓝斑核（LC）多巴胺含量显著减少，同时嗅束中影响神经发生的结构也受损，实验表明VMAT2的表达减少可以导致PD的非运动症状，如抑郁，嗅觉减退，而联合MPTP干预导致的黑质外多巴胺系统退行性病变及神经生发结构的受损可能是其潜在的机制。