膳食外泌体miR-23b对大骨节病软骨细胞损伤的保护作用及其机制

Previous studies have identified T-2 toxin and low selenium as suspected pathological factors of Kashin-Beck disease (KBD), however, the mechanism of cartilage injury of juvenile KBD caused by T-2 toxin and low selenium is still a conundrum. The most advanced international researches have established the protective or therapeutic role of dietary exosome miRNAs in repairing chondrocyte lesions by regulating the expression of genes and proteins which are associated with cellular apoptosis and extracellular matrix. Therefore, co-culture chondrocyte with exosome, gene interference and animal experiment would be applied in this study to explore and to establish the protective role of dietary exosome-miR-23b through PKA signaling pathway. The main contents are as follows: 1) to identify the relationship between the exosome and cartilage damage of KBD patients; 2) to identify the mechanism of miR-23b in cartilage damage through regulating PKA pathway; 3) to establish the therapeutic role of dietary exosome-miR-23b in repairing chondrocyte lesions induced by T-2 toxin and low selenium. To summarize, this study would establish the therapeutic role and mechanism of dietary exosome-miR-23b in repairing chondrocyte lesions on molecular, cellular and organic levels, which would promote the pathogenesis study and provide evidence to discover new biomarker and therapeutic means for KBD simultaneously.

针对我国大骨节病可疑致病因素粮食T-2毒素中毒和低硒特异性损害儿童软骨细胞机制未明及缺乏有效治疗靶点的关键科学问题，基于国际上膳食外泌体miRNA调控软骨细胞凋亡、细胞外基质相关基因及蛋白表达而控制软骨细胞损伤性疾病的最新研究进展，本课题拟采用外泌体软骨细胞共培养、基因干扰技术和动物实验，重点研究膳食外泌体miR-23b对大骨节病软骨细胞损伤的保护作用及其分子机制，主要研究内容有1）确定外泌体与大骨节病软骨细胞损伤的关系；2）明确miR-23b介导PKA通路在大骨节病软骨细胞损伤中的作用机制；3）明确膳食外泌体miR-23b对T-2毒素和低硒所致大骨节病软骨细胞损伤的保护作用。预期结果将明确膳食外泌体miR-23b对大骨节病软骨细胞损伤的保护作用途径，从组织、细胞和分子不同水平上确定大骨节病致病因素和损伤靶向干预作用，以推进大骨节病发病机制研究，为发现新的生物标志物和治疗手段提供科学依据。

针对我国大骨节病可疑致病因素粮食T-2毒素中毒和低硒特异性损害儿童软骨细胞机制未明及缺乏有效治疗靶点的关键问题，本项目基于外泌体miRNA调控软骨细胞凋亡、细胞外基质相关基因及蛋白表达而控制软骨细胞损伤性疾病的最新进展，结合血液、软骨细胞和组织外泌体miRNA测序、WB、qRT-PCR、IHC和生信分析等技术，获得主要研究结果：（1）大骨节病血清外泌体miRNA测序共鉴定出2771个miRNA，包含miR-23b在内的20个差异表达外泌体miRNA，其中4个上调和16个下调；（2）经qRT-PCR、WB和IHC检测证实，miR-23b在成人大骨节病软骨细胞中显著低表达，PKA信号通路关键分子PRKACB基因和蛋白在儿童和成人大骨节病软骨细胞和组织中均显著高表达；（3）大骨节病软骨细胞外泌体miRNA检测鉴定出1861个miRNA，包括53个差异表达外泌体miRNA，其中19个上调，34个下调；（4）大骨节病软骨细胞外泌体差异表达miRNA共同调控靶基因TIMP2、TLR3、ADAM12、PPARG和SMAD3，经CTD数据库查询表明上述基因均可与硒、亚硒酸钠、T-2毒素等发生交互作用，影响其mRNA和蛋白表达；（5）采用PLINK1.90对上述基因进行基于加性模型、显性模型和隐形模型的单位点关联分析，结果表明TIMP2基因rs4789936位点、TLR-3基因rs3775296位点、ADAM12基因rs1871054位点、PPARG基因rs12629751位点和SMAD3基因rs6494629位点与大骨节病显著相关；（6）大骨节病软骨细胞外泌体差异表达miRNA共同调控23种硒蛋白及其合成相关靶基因，且RT-PCR检测结果表明大骨节病软骨细胞中GPX3、DIO1、DIO2和DIO3基因在mRNA水平显著上调；（7）软骨细胞外泌体差异表达miRNA靶调控细胞周期蛋白及其依赖性激酶调控基因差异表达与大骨节病软骨细胞损伤相关；（8）结合双份饭法、HGAFS、CDGSS3.0膳食软件和因子分析明确病区儿童硒及综合营养现状和膳食结构缺陷。本项目研究结论为揭示外泌体差异表达miRNA参与大骨节病软骨细胞损伤提供了重要依据，同时为停止硒盐防治措施后如何巩固大骨节病防治效果提供支撑。受本项目资助已发表SCI论文7篇，中文核心期刊1篇，参加国内/际学术会议4人次并做大会报告3次。