MPTP诱导的人源化α突触核蛋白98基因修饰猕猴帕金森氏病模型的建立

The PD（Parkinson’s disease） animal model based on the gene-environment interaction is becoming the new direction, since the “multi-hit” theory of PD pathogenesis was proposed. a-synuclein, as the main component of lewy body, is closely related to the pathogenesis of PD, and its C-terminal truncate may be the key factor leading to the formation of lewy body. Our previous studies have shown that a-synuclein 98 (a C-terminal truncate was found increased express in the brain of PD patients) over-expressing dopaminergic cells were demonstrated significantly increased cytotoxicity and aggregate formation in presence of neurotoxicity agent. In this study, the humanized a-synuclein macaque was acquired (At the same time, the embryo library of the transgenic macaque was established), and the toxic agents-MPTP was injected in these macaques. Then these macaques were evaluated using the behavior, biochemistry and pathology methods (At the same time, the evaluation database of PD macaque was established). At last, the humanized gene animal disease model platform was established. Then a PD animal model with good construct validity, face validity and predictive validity, and caused by both a-synuclein 98 and MPTP, was generated and could be used to explore the disease mechanisms and drug screening.

自提出帕金森氏病（Parkinson’s disease,PD）发病的“多因素诱导”理论以来，应用基因-环境相互作用的方式来建立PD动物模型正成为发展方向。a突触核蛋白作为PD病理特征路易小体的主要成分，与PD的发病密切相关，并且其C短截短体可能是导致路易小体形成的关键因素。我们前期的研究表明，作为PD病人脑内高表达的a突触核蛋白C端截短体，a突触核蛋白98在其过表达的神经元内更容易聚集并对神经元产生毒性作用。因此，本研究拟采用CRISPR/Cas9介导的基因组定点编辑技术获得人源化的a突触核蛋白98基因修饰猕猴，同步建立其胚胎库，然后进一步用环境毒素MPTP诱导，进而通过行为学、生物化学以及病理学进行综合评价，同步建立模型评价指标数据库，最终建立人源化基因修饰灵长类动物模型平台，获得a突触核蛋白98和MPTP共诱导的具有良好建构、表面及预测效度的PD模型，用于疾病机理的研究及药物筛选。

通过该项目的实施，完成了非人灵长类动物的促排卵、卵细胞体外授精、胚胎的修饰、体外培养、冻存、复苏以及胚胎的受体猴移植等环节的优化，获得了大量的关键技术，成功建立了试管猴技术体系平台，并获得alpha-synuclein98怀孕的胚胎7个，出生7只子代猴，出生后死亡3只，目前存活4只，后续基因正在鉴定过程中。同时，我们还建立了MPTP猴模型的行为学评价体系。项目实施过程中，发表基金标注的文章8篇；并且，已将项目实施过程中的一些关键技术进行总结，并已申请相关专利2项。在项目实施过程中，1人获得在职硕士研究生学位，1人获得专升本学位，项目负责人马开利2016年入选云南省医学学科带头人培养计划，2018年入选云南省青年拔尖人才培养计划，2019年云南省技术创新人才培养出站并获得云南省技术创新人才证书，课题组成员杜廷福2019年入选云南省医学学科后备人培养计划。该平台体系的建成将为后续胚胎研究、辅助生殖研究以及非人灵长类疾病模型的建立等奠定重要的基础。