父代新生期七氟烷暴露后应激相关障碍及其跨代遗传：miR-29b靶向DNMTs调控KCC2/GABAAR的机制研究

新生期麻醉药物暴露是一种环境应激因素，可引起远期神经内分泌异常和情绪障碍。我们已发现KCC2甲基化及NKCC1/KCC2表达比例改变导致的GABAAR极性异常及其对HPA轴的抑制作用减弱是七氟烷对发育脑的潜在毒性机制。然而，仍不清楚介导KCC2甲基化的上游分子。我们首次提出miR-29b调控DNMTs介导的KCC2高甲基化是新生期七氟烷暴露后应激相关障碍的父系表观遗传机制。本项目拟1)通过TLDA测序及验证筛选目标miRNA; 2)运用质粒构建及双荧光素酶报告基因测定明确miR-29b和DNMT3a的结合; 3)运用工具药及构建AAV载体，进一步阐明miR-29b靶向DNMTs调控的KCC高甲基化在七氟烷发育期神经毒性中的分子机制。本项目不仅充分揭示新生期七氟烷暴露引起远期及子代应激相关障碍的分子机制，更可以为其他以应激反应紊乱为病理生理特征的神经精神疾病提供潜在的治疗靶标。

Neonatal anesthetic exposure is an environmental stress factor that could cause long-term neuroendocrine abnormalities and mood disorders. We have found that an increased degree of methylation of KCC2 and changes in the expression ratio of NKCC1/KCC2 induce abnormal GABAAR polarity and weakening of its inhibitory effect on the HPA axis, which is the potential mechanism of developmental neurotoxicity of sevoflurane. However, it is still unclear about the upstream regulatory molecules involved in the methylation of KCC2. We propose for the first time that miR-29b regulated DNMTs-mediated KCC2 hypermethylation acts as a paternal epigenetic mechanism of stress-related disorders after neonatal sevoflurane exposure. This project is intended to 1) screen and verify miRNA by TLDA sequencing; 2) define the binding of miR-29b and DNMT3a by plasmid construction and dual-luciferase reporter gene assay; 3) use drugs and construction of AAV vectors to further clarify the molecular mechanisms of miR-29b targeting DNMTs regulated KCC2 hypermethylation underlying the developmental neurotoxicity of sevoflurane. The project could not only fully reveal the molecular mechanism of long-term and offspring stress-related disorders caused by neonatal sevoflurane exposure, but also provide potential therapeutic targets for other neuropsychiatric diseases with disordered stress response system as pathophysiological characteristics.