多聚核苷酸激酶在小鼠神经系统发育过程中的作用

The nervous system is one of the earliest systems in the process of embryonic development. Many genes are necessary for brain's shaping and differentiation, Wnt family controlling cell proliferation and differentiation, mean while Bcl-2, P53 families are for apoptosis. Polynucleotide kinase 3'-phosphatase (PNKP) is the most important enzyme participating in the repairment of miscellaneous damaged nucleic acids, and it plays a very important role in maintaining genomic stability. Recently, it was found that mutation of PNKP is associated with early-onset, intractable epilepsy, delay development (MSCZ) directly, but researches about how PNKP inactivation leading to MSCZ in embryonic development is not reported. This study intends to mutant the mouse pnkp gene with CRISPR/Cas9 system, establishing MSCZ mouse development model. Based on immunofluorescence techniques, coIP- mass spectrometry, a sensitive label-free detection technology PNKP active, we plan to investigate the role of PNKP in the development of nervous system. In order to find out the roles of PNKP in the mouse nervous system during development mode, and clarify the mechanism of mouse embryonic nervous system development further, we will focus on the changes of neurons number, distribution, differentiation, apoptosis and signaling pathways related. It will throw light on the study of human neurodevelopment mechanism.

神经系统是胚胎最早发育的系统之一。发育过程既需要Wnt等家族基因参与的细胞增殖分化，也需要Bcl-2等基因家族所调控的细胞凋亡，实现脑组织的塑形与分化。多聚核苷酸激酶（PNKP）是各种核酸损伤修酶，在维持基因组稳定性方面有十分重要的作用。最新研究发现PNKP的突变、缺失与早发性、顽固性癫痫、小脑畸形、发育迟缓（MSCZ）直接相关。但是关于PNKP的失活如何在胚胎发育过程中导致MSCZ的研究尚未见报道。本研究拟利用CRISPR/Cas9 技术定点突变小鼠pnkp基因，建立小鼠MSCZ实验动物发育模型，通过免疫荧光技术、coIP-质谱技术、免标记PNKP活性灵敏检测技术，研究PNKP失活在神经系统发育过程中引起的脑神经元数量、分布、分化、凋亡等的变化以及有关的细胞信号通路。确定PNKP在小鼠神经系统发育过程中的作用模式，阐明小鼠胚胎神经系统发育的机理，可能为人类神经发育机理的研究提供新的思路。

多聚核苷酸激酶是一个非常重要的DNA修复因子，它主要参与了除了同源重组以外的所有修复过程。通过本项目的实施，使我们完善了多聚核苷酸激酶的重要生理学功能。多聚核苷酸激酶的缺失突变是致死性的突变。单碱基突变引起的多聚核苷酸激酶突变能够使得小鼠获得人MSCZ样症状，但是小鼠很快就会死亡。再一次说明多聚核苷酸激酶的发育重要过程。同事通本项目的实施我们建立了一种全新的基于芘分子激发体的单链DNA修复能力的直接评价方法，该方法灵敏直接，能直接判断细胞的DNA修复能力，为将来研究DNA修复相关的通路提供了基础。另外更重要的是在课题的支持下，迅速掌握的碱基编辑技术后，课题负责人获得了转基因重大专项课题的支持，使课题负责人能够迅速成长起来为今后的科学研究提供了桥梁作用。项目执行期间发表了SCI论文3篇，获得吉林省自然科学奖二等奖1项。