基因启动子区组蛋白甲基化修饰对后纵韧带骨化发生中的作用及机制研究

The ossification of posterior longitudinal ligament (OPLL) is one of the most common diseases in spine surgery department. Its onset may cause severe tetraparesis, urine and stool incontinence to the patients, so to prevent and cure such disease is an urgent matter. Although many have reported the factors that may cause such disease, however, the specific mechanism is not known. Under several previous funding of NSFC, our group has dedicated to find the core genetic factors that modulate the ossification process of the ligament cells. Although many unique core regulators were found, the initiation factors or the upstream regulatory mechanism of these genes were far from known. With the discovery of epigenetic factors in gene expression regulation, we have hypothesized that these epigenetic factors may have contributed to the initiation of core factors that cause OPLL. In this study, we take advantage of chromatin immunoprecipitation and sequencing technology to find the differentially expressed histone methylation modification sites in a global style. By analyzing the promoter region histone methylation level of core factors in OPLL and control ligament cells, we try to find the responsible methylation modification that initiates their expression, and also try to find new disease-related sites to expand the mechanism network of OPLL pathology.

后纵韧带骨化症（OPLL）是脊柱外科常见的疾病，造成严重的脊髓压迫后会导致四肢瘫痪和大小便失禁，是严重危害患者健康的疾病。尽管多项研究表明OPLL与遗传、代谢、环境等多种因素有关，但具体发病机制并不明确。课题组前期在连续多项国家自然基金的资助下发现了一批具有OPLL特异性的致病相关基因及调控分子，然而这些致病因子在OPLL中出现表达异常的原因仍未被揭示。近年来随着对表观遗传学认识的不断加深，人们认识到除了核酸等遗传物质以外还存在着其它的“遗传密码”能够影响基因的表达、细胞的表型甚至是个体的发育。因此课题组拟针对具有重要基因表达起始阶段调控作用的组蛋白甲基化修饰进行系统组学研究。利用染色质免疫共沉淀、高通量测序等手段对致病因子所在基因的组蛋白修饰情况进行分析和研究，同时通过生物信息学分析查找新的致病相关基因，最终通过研究组蛋白甲基化水平差异的机制来解释并完善致病基因出现异常的机理。

后纵韧带骨化症（OPLL）作为脊柱外科最为常见的疾病之一，由于其能够导致四肢瘫痪和大小便失禁，是严重危害患者健康的疾病。研究表明其致病因素较多，但具体的发病机制并不明确。近年来随着对表观遗传学认识的不断加深，人们认识到除了核酸等遗传物质以外还存在着其它的“遗传密码”能够影响基因的表达、细胞的表型甚至是个体的发育。因此课题组拟针对具有重要基因表达起始阶段调控作用的组蛋白甲基化修饰进行系统组学研究。利用染色质免疫共沉淀（ChIP）联合高通量DNA测序手段对韧带细胞基因的组蛋白修饰情况进行分析和研究，通过多组学手段联合分析，发现疾病特异性组蛋白甲基化特征及差异基因。通过偶联RNA测序结果构建差异基因调控网络，并找出潜在的致病关键基因，为后续进一步探索OPLL疾病的始动因素提供重要理论依据。通过针对该网络进行验证，明确miRNA-10a/ID3/RUNX2轴在后纵韧带细胞中参与成骨分化的作用及对OPLL疾病进程的影响，明确了致病相关miRNA通过细胞外囊泡的形式影响正常韧带细胞的TAK1、PBX1从而促进韧带细胞成骨分化的作用机制。相关内容明确了OPLL疾病的始动及促进因素，同时创新性地从微环境角度解析了OPLL疾病的发病机制，联合表观遗传学调控分子补充了现有OPLL疾病致病分子机制网络的不足，具有较好的创新性。构建了OPLL模型小鼠并验证了miRNA-181具有疾病靶向治疗的价值，为后续靶向治疗的开发奠定重要基础。通过对比细胞水平以及外周血中OPLL特异性非编码RNA成分，找到关键性miRNA可用于疾病诊断。相关内容以论著形式发表相关SCI期刊论著10篇，其中影响因子10分以上1篇，5分以上1篇，培养硕士研究生2名。相关成果获教育部科技进步二等奖，授权国家发明专利1项、国际发明专利1项，实用新型5项。