靶向诱导USP18基因甲基化对脑功能的影响及其在小儿脑性瘫痪发病中的意义

Cerebral palsy in children is a seriously disabling disease which threats to children's health. The etiology and pathogenesis of cerebral palsy have been research hotspot of epidemiology. The previous study, the methylation differential genes in normal-cerebral palsy identical twins were screened by whole genome sequencing chip. We for the first time found the hypermethylation of ubiquitin specific peptidase 18 (USP18) gene in the cerebral palsy twins. Previous studies have shown that USP18 gene is involved in interferon-induced antiviral response and is associated with brain injury. Therefore, we speculate that viral infection-induced by USP18 may be associated with the pathogenesis of cerebral palsy. Based on the speculation, the effects of inhibitive USP18 expression on the neurobehavioral and brain tissue injury in mice were investigated by using USP18 hypermethylation mice which were constructed by lentivirus-mediated transgenesis and shRNA-induced DNA methylation technique. The molecular mechanism of inhibitive USP18 expression on brain injury were studied by electron microscopy, immunohistochemistry and immunoblotting. This study has an important scientific theoretical significance for in-depth understanding of the pathogenesis of cerebral palsy, and can provide a deep theoretical basis for prevention and treatment of cerebral palsy.

小儿脑性瘫痪是严重威胁儿童健康的致残性疾病，其病因及发病机制是流行病学研究的热点。本课题组前期采用全基因组测序芯片筛查正常-脑瘫同卵双生子甲基化差异基因，首次发现泛素特异性蛋白酶18（USP18）基因在脑瘫患儿中呈现高甲基化状态。以往研究证明该基因参与干扰素诱导的抗病毒反应，并与脑功能损伤关系密切。因此，我们推测USP18介导的抗病毒反应可能与脑瘫发病有关。基于此推测，本项目继续针对脑瘫双生子人群，开展病毒感染史调查，明确病毒感染与USP18甲基化及脑瘫发病的关系；采用慢病毒结合shRNA诱导DNA甲基化技术，构建USP18高甲基化小鼠模型，研究USP18表达抑制对小鼠神经行为及脑组织的损伤作用；采用电子显微镜、免疫组化、蛋白印迹等技术，探究USP18表达抑制引起脑组织损伤的分子机制。此项研究对于深入了解脑瘫的发病机制具有重要的科学理论意义，同时可为脑瘫的科学防治提供深层次的理论依据。

小儿脑性瘫痪是严重威胁儿童健康的致残性疾病，其病因及发病机制是流行病学研究的热点。本课题组前期采用全基因组测序芯片首次发现泛素特异性蛋白酶18（USP18）基因在脑瘫患儿中呈现缺失表达状态。我们推测USP18介导的抗病毒反应可能与脑瘫发病有关。基于此推测，本项目继续针对脑瘫双生子人群，开展病毒感染史调查，明确病毒感染与USP18表达及脑瘫发病的关系；采用慢病毒包装载体注射和条件性基因敲除，分别构建USP18基因敲减和USP18基因敲除小鼠模型；采用旷场实验、电子显微镜、免疫组化、蛋白印迹等一系列技术手段，探究USP18表达缺失与小鼠行为和脑功能损伤的关联和作用机制。流行学研究发现了一系列脑瘫发病风险因素，例如双生子母亲有人工流产史[OR=10.001(2.201-45.450)]、母亲有病毒性疾病感染史[OR=4.065(1.234-13.393)]、母亲被动吸烟[OR =21.561(4.046-114.897)]、产时孕周≤34周[OR=9.821(2.905-33.204)]、母亲孕期产前未进行全身检查[OR =10.001(2.201-45.450)]等；成功建立了USP18基因敲减小鼠模型和USP18基因敲除小鼠模型；机制研究表明，USP18基因表达缺失可以引起小鼠行为异常、脑部发育异常以及神经元功能损伤，同时发现小胶质细胞活化和细胞凋亡事件与USP18基因表达缺失引起的脑功能损伤具有关联性。此项研究对于深入了解脑瘫的发病机制具有重要的科学理论意义，同时可为脑瘫的科学防治提供深层次的理论依据。