LFA-1/Syk交互协同RANKL/NF-κB信号通路调控破骨细胞分化的机制研究

After artificial joint replacement, periprosthetic osteolysis is a difficult problem to be solved by the surgeons, and osteoclasts are closely related. However, the molecular mechanism that regulates the differentiation of osteoclasts is not yet completely clear. Although we have demonstrated that a variety of drugs can regulate the differentiation of osteoclasts through the classical RANKL signaling pathway(Cell Physiol Biochem and Int Immunopharmacol, 2017 ), but that is not the only one. We have found that the lymphocyte functional related antigen-1 (LFA-1) in the integrin family can regulate the differentiation and maturation of osteoclasts through the Sky signaling pathway. However, it is not clear that the specific regulation mechanism, and the interaction between the classical RANKL/NF-κB signaling pathway are still unclear. In order to explore this scientific problem, the following researches are to be carried out in this project: 1. Further clarifying the molecular mechanism of LFA-1 regulation of osteoclast differentiation through Syk signaling pathway; 2. To elucidate the interaction between LFA-1/Syk and RANKL/NF-κB signaling pathway in the regulation of osteoclast differentiation; 3. Establishment of an animal model for further validation studies in vivo. This research is expected to provide a new therapeutic target for treating and preventing osteolysis diseases.

人工关节置换术后假体周围骨溶解是外科医生亟待解决的难题，其发生与破骨细胞密切相关。然而调控破骨细胞分化的机制尚不完全明确。虽然我们研究证实多种药物可通过经典的RANKL途径调节破骨细胞的分化(Cell Physiol Biochem与Int Immunopharmacol, 2017 )，但其不是唯一通路。我们预实验发现整合素家族中的淋巴细胞功能相关抗原-1（LFA-1）可通过Sky信号通路调控破骨细胞的分化。然而其具体机制、及是否与RANKL/NF-κB信号通路之间存在交互调控作用尚不明确。为了探讨该科学问题，本项目拟开展以下研究：1.明确LFA-1经Syk信号通路调节破骨细胞分化的机制；2. 阐明LFA-1/Syk与RANKL/NF-κB信号通路在调控破骨细胞分化中的交互调控情况；3.构建骨溶解动物模型，在体内对相关机制进一步研究。本研究成果有望为骨溶解病的防治提供一个新治疗靶点。

在本研究项目中，我们完成了临床骨质疏松病人的临床标本中LFA-1和Syk表达量，Syk磷酸化水平检测，共表达检测；通过qPCR检测LFA-1和Syk的mRNA水平；WB检测LFA-1和Syk的蛋白质水平，以及Syk的磷酸化水平；IHC检测LFA-1和Syk的蛋白质水平，以及Syk的磷酸化水平；以及完成了敲低LFA-1和/或抑制p-Syk (抑制剂：Piceatannol)对分化的破骨细胞的影响研究。通过TRAP染色，Control组（未分化组）和RANKL+CSF1处理（破骨细胞分化组）组，计数多核细胞的细胞数，并通过Pit Formation Assay检测BMM分化为破骨细胞后的骨吸收能力，以及检测Control组和RANKL+CSF1处理组BMMs中LFA-1和Syk、p-Syk的共表达情况；基于此检测结果，我们通过WB检测了各分组中LFA-1的表达，ICAM-1的表达，Syk的表达，以及p-Syk的表达情况，以及通过qPCR检测破骨细胞阳性标记物如TRAP，DC-STAMP，Cathepsin K，OSCAR，c-Fos，NFATc1的表达水平和通过WB检测上述阳性标记物的表达水平。.本研究项目共发表SCI论文11篇，单篇最高6.384分，影响因子合计47.582，中文核心期刊3篇。包括《Inhibition effects of a natural inhibitor on RANKL downstream cellular signalling cascades cross-talking》，《Integrin-associated molecules and signalling cross talking in osteoclast cytoskeleton regulation》，《Regulation effects of melatonin on bone marrow mesenchymal stem cell differentiation》。在破骨细胞分化研究方面达到了国内外先进水平，具有较高的创新性。