软骨形成和发育过程中Sox9基因转录调控机制研究

At present, there are more than 400 million osteoarthritis patients worldwide, of which more than 100 million patients are in China. Osteoarthritis is mainly caused by cartilage injury. Due to the poor self-healing and regeneration capacities, there are still no suitable treatments yet available. Therefore, the treatment of osteoarthritis can be only achieved by discovering ways of cartilage repair and regeneration. Sox9, a pivotal transcription factor, plays an important role in regulating chondrocyte generation, proliferation and differentiation. It has been shown that the temporal, spatial, and quantitative expression of Sox9 must be precisely regulated to ensure proper development and adult maintenance of cartilage. We systematically screened the upstream region of Sox9 and newly identified several enhancers which had strong, overlapping activity in developing and adult cartilage (Yao, et al., Nucleic Acids Res). In this study, in order to demonstrate the mechanism of transcriptional regulation of Sox9 in cartilage, we will identify transcription factors and signaling pathways involved in the transcriptional regulation of Sox9 gene in cartilage by biotin-streptavidin purification system, mass spectrometry technology and gain (loss)-of-function assay. These findings will provide theoretical guidance and novel target for the treatment of osteoarthritis.

目前全世界骨关节病患者超过4亿，其中我国患者多达1亿以上。骨关节疾病主要由软骨损伤引起，由于软骨组织再生能力差，目前仍然没有治疗骨关节疾病的有效方法。因此，治疗骨关节疾病必须找到实现软骨修复和再生的方法。Sox9是调控软骨细胞产生、增殖和分化的重要转录因子，Sox9 基因在时空上的准确转录是软骨形成和发育的关键环节。我们在Sox9基因上游鉴定出调控Sox9基因在不同分化阶段软骨组织中表达的增强子（Yao, et al., Nucleic Acids Res, 2015）。本课题通过生物素-链霉亲和素分离系统、质谱分析技术及功能获得和缺失实验等手段，鉴定参与Sox9基因在软骨组织中转录调控的转录因子和信号通路，揭示Sox9基因在软骨组织中的转录调控机制，这些研究结果将为治疗骨关节疾病提供理论指导和治疗靶点。

本课题通过生物素-链霉素 (Biotin-Strepavidin) 亲和系统分离纯化方法，结合质谱鉴定技术，从软骨细胞中分离并鉴定出多种与Sox9 增强子结合的转录因子。针对这些转录因子进行进一步筛选，筛选出12种与软骨生长发育密切相关的转录因子，主要包括Runx3、Trps1、Hdac2、Nfib和Nfix等。通过真核表达载体构建、细胞转染、qRT-PCR、western blot、细胞染色、报告基因活性检测及转录组学分析等手段和方法，系统深入研究了这些转录因子对软骨细胞增殖、分化及基质合成等影响，同时观察了这些转录因子对软骨细胞基因表达水平，尤其是Sox9及下游靶基因的调控作用。研究结果表明转录因子Runx3和Hdac2对软骨细胞增殖及基质合成具有负调控作用，而对软骨细胞分化具有正调控作用；转录因子Nfib、Nfix和Trps1对对软骨细胞增殖及基质合成具有正调控作用，而对软骨细胞分化具有负调控作用；这些调控作用主要是通过与Sox9软骨增强子结合，进而调控Sox9及其下游靶基因Col2a1和Acan等来是实现的。因此，本研究阐明了基于Trps1/Nfib/Nfix-Sox9增强子-Sox9转录调控-软骨细胞增殖分化正向调控及Runx3/Hdac2-Sox9增强子-Sox9转录调控-软骨细胞增殖分化负向调控的Sox9信号调控网络。这些研究结果为治疗骨关节疾病提供理论指导和治疗靶点。通过本课题的实施，发表SCI论文7篇，培养博士研究生2名，硕士研究生3名。接下来的研究工作中，我们将进一步采用基因敲除及多组学联合技术等对这些转录因子的功能进行更加深入的研究。