PI3K/Akt信号途径在调控大骨节病软骨细胞自噬及代谢中的作用

Previous studies have found that autophagy is defective and related to metabolic disorder in chondrocytes with Kashin-Beck disease (KBD). The mechanism, that environmental risk factor (T-2 toxin) causes changes in chondrocyte function, is unknown. To solve the above issues, this project plans to study PI3K/Akt signaling pathway that regulates autophagy and metabolism in KBD chondrocytes, combining result of genetic data analysis and basing on study design, "environmental factors - differently expressed genes - signaling pathways - cellular effects", using RNA interference, gene chip technology, metabolomics and molecular biotechnology. The main contents consist of: 1) the characteristic of Akt expression in KBD cartilage and chondrocytes that will be defined; 2) autophagy-related and metabolism-related genes which are regulated by Akt will be screened in KBD chondrocytes, and gene pathways and networks will be built; 3) the regulation and possible molecular mechanisms of PI3K/Akt signaling pathway in relation to autophagy and metabolism in KBD chondrocytes will be determined; 4) the mechanism involved in damage of chondrocytes function, due to T-2 toxin, via PI3K/Akt signaling pathway will be defined. Expected results will demonstrate the regulation of PI3K/Akt signaling pathways to autophagy and metabolism in KBD chondrocytes. It will be in valuable to pathogenesis studies and targeted treatments in KBD.

针对前期研究发现大骨节病软骨细胞自噬功能缺陷及其影响软骨细胞代谢的发病机制，以及环境危险因素T-2毒素引发软骨细胞功能改变机制不明的问题，本课题结合基因数据分析结果，遵循“环境因素-差异基因-信号途径-细胞效应”的研究思路，拟采用基因干扰、基因芯片、代谢组学和分子生物技术等，重点研究PI3K/Akt信号途径在调控大骨节病软骨细胞自噬及代谢中的作用。主要研究内容有：1）明确Akt在大骨节病软骨组织和软骨细胞中的表达特点；2）筛选大骨节病软骨细胞中Akt调控的自噬和代谢功能相关基因并构建基因通路和网络；3）确定PI3K/Akt信号途径调控大骨节病软骨细胞自噬和代谢的作用效应及可能的分子机制；4）确定T-2毒素通过PI3K/Akt信号途径损伤自噬和代谢等软骨细胞功能。预期结果将阐明PI3K/Akt信号通路对大骨节病软骨细胞自噬和代谢的调控作用，推进大骨节病发病机制研究和靶标治疗依据。

针对大骨节病发病的基因调控机制不明和软骨细胞功能自噬等功能改变的问题，本课题依据“大骨节病诊断标准”，收集了7例大骨节病和5例正常软骨组织，制作了组织标本并培养了软骨细胞，同时利用C28/I2软骨细胞系，免疫组织化学、免疫印迹、 qRT-PCR、基因干扰和基因芯片等技术，获得的主要研究结果有：①大骨节病软骨组织和软骨细胞中Akt1基因和蛋白高表达；②基因干扰技术成功使得软骨细胞中Akt1基因沉默或高表达；③利用嘌呤霉素筛选并建立了Akt1基因干扰之后的稳定表达株；④自噬基因ATG4C的多态性位点rs11208030、rs4409690、rs12097658和rs6587988与大骨节病患病风险显著相关，实验确认自噬相关基因ATG4C在mRNA和蛋白水平上在大骨节病和正常关节软骨中表达存在显著差异。⑤筛选出了大骨节病软骨细胞差异表达的mRNA和lncRNA，探讨了差异表达的lncRNA以及共表达的mRNA可能发挥的基因调控途径。本项目资助硕士研究生1名，发表本领域高水平SCI论文2篇，影响因子分别是5.454和4.122，中文期刊论文3篇。