CRISPR/Cas介导的GCPII及III基因联合剔除启动脑损伤后内源性神经保护的研究

Our previous studies have demonstrated the neuroprotective effects of NAAG peptidase inhibitor and GCP II gene knockout (KO) after traumatic brain injury (TBI). On the basis of the compensatory phenomenon of GCP III expression, in this study, we plan to exploit the GCP II and III double KO strategy mediated by CRISPR/Cas system to generate a brand new KO mice line with improved efficiency of NAAG peptidase inhibition. Moreover, we will use the lateral controlled cortical impact (CCI) TBI model in combination with the in vivo microdialysis technique to provide new insights into the characteristics of the cell death patterns, neurotransmitter release and neuroprotective signal transduction after TBI. We also will evaluate whether the GCP II/GCP III double KO mice appear to be less susceptible to TBI as compared to the wild-type (WT) mice or the single gene KO mice. This study will expand the understanding of the biochemical characteristic of the new target GCP III and brain’s natural endogenous neuroprotective system of controlling glutamate excitotoxicity by the GCP II/GCP III double KO and further supports the hypothesis that blockade of NAAG peptidases may be a promising potential therapeutic strategy for TBI.

在前期研究发现NAAG肽酶抑制剂及GCP II基因剔除对创伤性脑损伤具有保护作用并观察到GCPIII “补偿性表达现象”基础上，拟利用最新的CRISPR/Cas基因剔除技术在国际上首次尝试构建GCP II/GCP III基因联合剔除小鼠，进一步提高NAAG肽酶阻断效率；同时结合应用“控制性脑皮质损伤模型”及在体微透析技术，通过与野生型小鼠比较，观察从分子水平完全阻断NAAG肽酶两种亚型活性对脑损伤后突触间隙递质释放调控、神经保护信号转导、细胞死亡模式等多个环节的影响，重点比较GCP II/III基因单独剔除及联合剔除启动内源性神经保护的不同特点。本研究有利于进一步阐明GCPIII新靶标的生化特性及GCP II/III肽酶双抑制新模式从源头上阻断谷氨酸兴奋毒性级联反应从而提高小鼠创伤性脑损伤耐受性的确切疗效及分子机制，揭示继发性脑损害过程的“内源性神经保护机制”，为颅脑创伤治疗提供新策略。

既往本课题组发NAAG肽酶抑制剂及GCP II基因剔除对创伤性脑损伤具有保护作用并观察到GCP III“补偿性表达现象”，因此，利用最新的CRISPR/Cas基因剔除技术首次成功构建了GCP II/GCP III基因联合剔除小鼠。结合应用“控制性脑皮质损伤模型”及在体微透析技术，我们发现GCP II/III基因联合敲除小鼠比GCP II/III基因单独剔除小鼠能更好的启动内源性神经保护效应，减轻神经元坏死和细胞凋亡，并改善损伤后小鼠的学习记忆能力。这阐明了GCPIII新靶标的生化特性及GCP II/III肽酶双抑制新模式从源头上阻断谷氨酸兴奋毒性级联反应从而提高小鼠创伤性脑损伤耐受性的确切疗效，进一步为颅脑创伤治疗提供了新策略。