大骨节病环状RNA表达、功能及其与T-2毒素的关系

The key scientific issues on the etiology, pathogenesis and early prevention of Kashin-Beck disease(KBD) to serious damage of population health in the endemic areas of China, the relationship between T-2 toxin and molecular markers of KBD and their application will be mainly studied by using the unique disease resources of KBD in China, the epidemiological methods and the modern molecular biology techniques, the main contents are: (1)Based on the abnormal CircRNA gene from KBD recently obtained by the high-throughput detection technology, using modern molecular biological techniques to determine CircRNA differentially expressed relationship with the occurrence and development of kashin-beck disease, evaluate the sensitivity and accuracy of the diagnosis for kaschin-beck disease；（2）The time and dose-response relationship during the damage chondrocyte induced by the T-2 toxin and molecular markers of KBD are investigated using the chondrocyte model, to provide a scientific basis to clarify the external enviroment-the internal enviroment-the molecular mechanism of cartilage damage.(3) Expand the samples based on the study of molecular markers above, to verify the CircRNA biomarkers with quantitative real-time PCR analysis, and confirm the clinical significance of CircRNA biomarker express with KBD diagnosis and prognosis . Invent a CircRNA potential biological marker kit for early screening of kaschin-beck disease, to enhance the level of early diagnosis, evaluation of treatment efficacy and prognosis and their clinical transformation for KBD in China.

针对严重危害我国病区人口健康的大骨节病病因、发病机制及早期防治的关键科学问题，利用我国特有的大骨节病疾病资源，采用人群流行病学调查和现代分子生物学技术，集中研究大骨节病分子标志物及T-2毒素对其影响作用，主要内容有：（1）基于高通量检测技术所获得大骨节病环状RNA（CircRNA）基因，利用现代分子生物学技术，确定CircRNA差异表达与大骨节病发生发展的关系，评价其诊断大骨节病的敏感性及准确性；（2）采用软骨细胞模型，观察T-2毒素与分子标志物在细胞损伤中的关系，为阐明大骨节病外环境-内环境-软骨损伤的分子机理提供科学依据；（3）基于上述研究，扩大样本进行分子标志物CircRNA的qPCR验证，确定CircRNA标志物表达与大骨节病及预后的临床指导意义，开发一款大骨节病CircRNA潜在生物学标志试剂盒用以早期筛查大骨节病，提升大骨节病早期诊断、治疗和预后判定的技术支撑和临床转化水平。

项目背景: 大骨节病（Kashin-Beck Disease，KBD）是我国特有的一种地方性、慢性变形性骨关节病。随着现代分子生物学的发展，KBD的发病机制已经深入到研究易感基因及环境反应基因的程度。我国目前KBD的防控缺乏早期检测指标，无法进行早期诊断与治疗。CircRNA具有组织特异性和稳定性的特点，已经证实其与OA的发生发展相关。本研究基于高通量检测技术获得KBD及OA相关CircRNA，为靶向治疗提供理论依据。.主要研究成果：应用CircRNA芯片技术检测KBD、OA患者和正常对照的软骨细胞差异表达CircRNA，应用qRT-PCR技术对显著差异基因在软骨细胞层面进行验证，进而扩大样本在外周血中进行临床验证。筛选出3个生物标志物：hsa-circRNA-0057421、hsa_circ_0032131、hsa_circ_0020014，建立了微创、快速、早期的基因诊断思维。.关键数据: 应用高通量技术筛选出KBD相对于正常对照差异表达CircRNA基因共886个，其中上调表达基因348个，下调表达基因538个；OA相对于正常对照差异表达CircRNA基因1380个，215个上调表达，1165个下调表达；KBD与OA组差异表达基因共1627个，上调表达基因1328个，下调表达基因299个。1）KBD与正常对照软骨细胞中筛选的差异表达基因hsa_circ_0057421_CBC1，应用ROC曲线对其诊断价值进行评估，曲线下面积（0.718，p<0.01），表明hsa_circ_0057421_CBC1区分KBD和健康对照有较高的灵敏度。2）OA患者的CircRNA基因表达谱不同于正常对照，应用qRT-PCR技术对hsa_circ_0032131的诊断价值进行评估，ROC曲线下面积（AUC）：0.8455，p<0.01，表明hsa_circ_0032131_CBC1区分OA患者和健康个体有较高的灵敏度。3）KBD与OA软骨细胞中筛选的差异表达基因hsa_circ_0020014_CBC1，应用ROC曲线对其诊断价值进行评估（曲线下面积0.6415，P<0.01），表明hsa_circ_0020014_CBC1具有潜在的鉴别诊断价值。.科学意义：采用高通量分析方法，探讨了CircRNA在原发性OA和KBD发生发展中的生物学功能，提高我国骨关节疾病的防控和监测水平。