HS6ST2调控软骨基质稳态参与OA发生发展的作用机制研究

Osteoarthritis (OA) is a most common degenerative joint disorder and is characterized by degradation of articular cartilage. The catabolism and anabolism imbalance of cartilage matrix is a key factor in the complicated pathogenesis of OA. Our previous work showed that HS6ST2, an enzyme involved in the transfer of sulfate to heparan sulfate, is downregulated in damaged cartilage tissues of OA patients and mediates matrix degradation, but little is known about its regulatory mechanism and its role in cartilage injury of OA. .Several experiments were designed for this project. Firstly, we plan to set up the DMM model in the Hs6st2 conditional knockout animals and determine the role of HS6ST2 in cartilage matrix metabolism and injury preliminarily. Secondly, based on the gain or loss of HS6ST2 with adenovirus or siRNA in chondrocytes, the molecular mechanism of regulation in cartilage metabolism mediated by HS6ST2 will be determined by means of gene expression analysis, signal pathway analysis, histological analysis and protein interaction analysis. Moreover, in order to determine the regulatory mechanism of HS6ST2 in cartilage lesion, we plan to ascertain the regulation in transcriptional level with dual-luciferase system and ChIP, and in post-transcriptional level with alternative splicing、ncRNA target relationship and analysis of protein modification. Eventually, to identify the role of matrix metabolism balance mediated by HS6ST2 in cartilage injury of OA, we plan to set up the DMM model in the Hs6st2 conditional knockout animals, and design the intervention with exogenous Hs6st2 rescue and downstream or upstream regulatory elements of HS6ST2. .In summary, function of HS6ST2 in matrix metabolism of cartilage and the pathological course of OA not only provide new insights into the role of sulfotransferase in matrix metabolism regulation, but also raise possibilities to intervene HS6ST2 for developing a novel therapeutic strategy of OA.

软骨基质代谢失衡是骨关节炎（OA）的重要病理特征。我们发现：细胞硫酸乙酰肝素修饰酶-HS6ST2在OA软骨中表达显著降低，且使基质分解代谢增强，但具体作用及调节机制不详。本研究首先在Hs6st2软骨特异敲除小鼠，通过DMM建立OA模型，检测关节软骨的病理改变，确定HS6ST2在OA发生发展中的作用；继而在原代软骨细胞中干预HS6ST2表达，通过分析基因表达、信号通路、相互作用蛋白和组织学观察等手段，确定HS6ST2调控软骨基质代谢平衡的分子机制；利用荧光素酶报告基因系统、染色质免疫共沉淀、可变剪接调控、ncRNA靶向分析、蛋白修饰检测等手段，确定转录和转录后水平调控HS6ST2表达的分子机制；最后利用Hs6st2条件敲除小鼠干预HS6ST2上下游重要调控因子的表达，明确HS6ST2介导的软骨基质代谢失衡的分子机制。结果将丰富对OA发病机制认识，为寻找新型高效生物标记和治疗靶点奠定基础。

骨性关节炎（OA）是一种常见关节退行性病变，软骨基质代谢失衡是OA软骨病变的成因之一，但是具体调控机制不明。我们前期研究发现HS6ST2在OA软骨中表达降低，但是具体功能不详。本项目提出了“HS6ST2 通过调控软骨基质代谢平衡进而参与 OA 软骨损伤”的假说，并从以下几方面研究进行了论证。首先分别检测OA患者和小鼠DMM模型的软骨组织中的HS6ST2表达，证实相比正常软骨组织，损伤软骨组织中HS6ST2存在明显的表达抑制现象；再利用Hs6st2条件敲除动物在体干预HS6ST2表达，证明HS6ST2缺失会加剧DMM手术造成的软骨基质丢失，证明HS6ST2存在软骨保护作用，利用Micro-CT分析证实HS6ST2缺失显著影响软骨下骨的重塑；接着分别利用3D微团培养和平面培养的原代软骨细胞实现HS6ST2的缺失与获得实验，证实HS6ST2可明显抑制软骨分解代谢基因表达，并增加整体软骨细胞基质含量，且两种HS6ST2转录本可以发挥类似的效果；最后利用RNA深度测序进一步分析HS6ST2对于软骨细胞基质代谢以及细胞稳态的影响，揭示了HS6ST2参与调控下游包括细胞外基质平衡、细胞周期、炎症信号、细胞凋亡、细胞通信、蛋白质翻译与修饰等多个信号通路。本研究明晰了HS6ST2在OA发生发展中发挥的重要作用，为OA发病机理的阐明、早期OA临床治疗策略以及靶向药物的研发提供了理论基础和实践证据。