环状RNA mmu_circ_0001724在推拿治疗脑瘫中的作用与机制研究

Recent studies suggest that circular RNA (circRNA) may play an important role in the occurrence of cerebral palsy (CP). Our previous studies showed that the target circRNA (mmu_circ_0001724) was downexpressed in white matter of rat CP model, and its overexpression by tuina effectively intervened CP. According to these studies, this project proposes that there is a new mechanism for the regulation of circular RNA in treatment of cerebral palsy with tuina. We will observe the changes of CP syndromes in rat models and oligodendrocyte changes such as cell proliferation in cell models to clear the function of circRNA in CP by using the overexpression of the target circRNA in lentivirus vector. Experimental methods such as RIP and luciferase reporter gene experiment etc will be used to clarify the expression and regulation of miRNAs and downstream mRNAs of the miRNAs adsorbed by the target circRNA, and to determine the regulatory relationships among target circRNA, miRNA and mRNA, and further clarify new mechanism of miRNA sponge of circRNA regulating CP. Blood samples from patients with CP will be obtained to test the function of the target circRNA in order to find CP biomarker. This project aims to clarify a new regulative mechanism of circular RNA in treatment of cerebral palsy with tuina and to provide a novel strategy for preventing and treating CP.

新近研究提示：环状RNA（circRNA）可能在推拿治疗脑瘫中具有重要调控作用。我们前期发现，目的circRNA（mmu_circ_0001724）在CP大鼠脑白质中表达下调，推拿上调其表达并有效干预CP。本项目据此提出：推拿治疗脑瘫中可能存在环状RNA调控新机制。拟对CP大鼠和少突胶质细胞模型进行目的circRNA过表达慢病毒转染，推拿干预CP，观察CP特征及细胞增殖等变化，明确目的circRNA调节CP新功能；用RIP和荧光素酶报告基因实验等明确目的circRNA吸附的miRNA及其下游靶基因表达与调控，探明目的circRNA、miRNA与mRNA之间调控关系，深度阐明目的circRNA调节CP的miRNA海绵新机制；取CP患者血标本验证目的circRNA，确立CP生物标志物。阐明目的circRNA在推拿治疗脑瘫中的作用和机制，为确立防治CP的新策略提供研究基础。

小儿脑性瘫痪（CP）致残性率高，严重危害小儿健康，给家庭及社会造成沉重的经济和精神负担，而CP发生机制尚未完全阐明，有效防治CP也极其欠缺。探明CP发生机制及确立有效防治CP策略，是目前研究的难点。本项目通过建立CP幼鼠模型，进行推拿干预，观察CP幼鼠脑白质的病理和行为学变化，检测circ_0001724表达情况；建立缺血缺氧少突胶质细胞模型，构建circ_0001724 慢病毒载体，并转染细胞，采用CCK-8法检测细胞的增殖能力，用transwell方法检测细胞的迁移能力，明确circ_0001724对细胞增殖和迁移能力的影响；通过生信分析技术预测和RIP-PCR实验验证，探研circ_000172海绵吸附的miRNAs，以及miRNAs调控的下游靶基因。研究发现，推拿可以促进CP幼鼠生长发育，提高CP幼鼠学习记忆和运动平衡能力；推拿可以降低CP幼鼠脑白质内髓鞘的内外径比值，促进受损髓鞘的修复；推拿可以通过调控circRNA_0001724表达，促进缺血缺氧少突胶质细胞的迁移能力和增殖能力，增强细胞活力，逆转CP病理变化。本研究采用动物行为学的方法进一步验证了推拿对CP幼鼠良好的康复治疗作用，用分子生物学的方法观察到CP幼鼠康复过程中脑白质的病理变化、目的环状RNA0001724表达的变化情况，阐明了circ_0001724海绵吸附的miRNAs和miRNAs调控的下游靶基因，为临床进一步推广应用中医推拿治疗CP提供理论依据，并对探索CP早期诊断生物标记物奠定实验基础。以该项目为依托，公开发表论文5篇，均标注有“国家自然科学基金资助（编号81860886）”，获实用新型专利授权3项，获云南省科技进步二等奖1项。培养硕士研究生5名，参加国内外相关学术交流16人次。