CXCL13/FAK对激素性股骨头坏死中MSCs转归的调控机制研究

It is a vital cytological event in the early stage of steroid-induced ONFH (osteonecrosis of femoral head，ONFH) that MSCs (mesenchymal stem cells) crisis could be induced by disordered microenvironment, whereas the underlying mechanism is still unknown as well as the key molecules. In recent researches, CXCL13 has been identified as a determinant of the fate of adult stem cells, moreover, its downstream molecule, FAK (focal adhesion kinase), is also a important survival signaling which acts as a messenger to link up multiple signaling pathways for the stabilization of microenviroment. Previously, we have confirmed that CXCL13 (chemokine CXC ligand 13) involved in the regulation of osteogenic microenvironment by affecting important cellular behaviour including homing, adhesion and survival, etc. Preliminary experiments also support the relevance among CXCL13, FAK and steroid-induced ONFH. On the basis of these findings, CXCL13/FAK signals are speculated as key candidates to regulate MSCs fate in the microenvironment of steroid-induced ONFH. So far, there are no detailed evidences of CXCL13/FAK effection in this particular microenviroment as well as their underlying molecular mechanism. We attempt to uncover the relationship among CXCL13/FAK signaling pathway, MSCs fate, and ONFH microenvironment. The regulating effects will be explored by cytological detection with MSCs labeling, tracing and imaging in vivo. In the meantime, proteome and CHIP techniques will be utilized to clarify the major function, regulating pattern and key molecule. Ultimately, it is expected to identify a novel target to repress early steroid-induced ONFH.

MSCs转归危象是激素性股骨头坏死（ONFH）的早期关键事件之一，其调控机制尚未阐明。国内外文献表明，微环境是影响MSCs命运的决定性因素；但是，它调控MSCs转归的关键分子尚不清楚。新近报道，CXCL13对微环境稳定与MSCs转归有重要贡献，其下游FAK也是重要生存信号，并可能作为信使分子衔接多个微环境调控通路。课题组已证实，CXCL13参与成骨微环境调控，指导MSCs归巢、黏附、生存等重要行为；预实验支持CXCL13、FAK与ONFH存在密切相关。我们推测：在ONFH微环境中，CXCL13/FAK是调控MSCs转归的关键分子。为证实这一假说，同时探索其确切机制，课题组将利用示踪标记、活体成像等技术，观察CXCL13/FAK对MSCs转归的影响；借助CHIP、蛋白质组方法，阐明其调控途径与主要机制。本研究有望进一步揭示ONFH病因机制，为早期阻遏ONFH提供新思路。

间充质干细胞（MSCs）转归危象是激素性股骨头坏死（ONFH）的早期关键事件之一，而微环境是影响 MSCs 命运的决定性因素，文献报道CXCL13对微环境稳定与MSCs转归有重要贡献，其下游 FAK 也是重要生存信号，并可能作为信使分子衔接多个微环境调控通路。因此,本研究的目的是探索在ONFH微环境中，CXCL13/FAK调控 MSCs转归的机制。我们成功建立了激素性ONFH细胞模型，发现早期ONFH MSCs中CXCL13和CXCR5的表达高于塌陷期和空白组，塌陷期MSCs凋亡显著上升，FAK沉默抑制MSCs增殖并上调凋亡基因Caspase-3和下调 Akt, NF-kb 和 Bcl-2的表达，从而促进MSCs凋亡。Lentivirus-CXCL13转染MSCs CXCL13高表达后可上调FAK的表达和磷酸化，促进MSCs的定向迁移。激光共聚焦显微镜观察到CXCL13高表达MSCs伸出更多伪足，与邻近细胞形成更紧密的突触联系，细胞间连接更优，细胞运动相关的骨架与微丝结构表现更为活跃。粘附蛋白（Laminin和E-Cadherin）和趋化调控分子（GRB2、RAS、ERK1/2）的表达均显著升高。同时我们也成功建立了激素性ONFH动物模型，发现早期激素性ONFH中CXCL13/CXCR5表达上调，而塌陷期激素性ONFH中CXCL13/CXCR5表达下调，CXCL13过表达MSCs移植可使股骨头坏死区高表达CXCL13和FAK蛋白,诱导更多的MSCs迁移并定植于股骨头坏死区，增强局部成骨，促进股骨头坏死的修复。本研究证实了CXCL13/FAK与早期激素性股骨头坏死（ONFH）的相关性，发现了CXCL13/FAK可通过调控MSCs的迁移、增殖、凋亡、粘附等影响其转归，并筛选出涉入其中的关键分子和信号通路，从而进一步阐明激素性ONFH的发病机制，为ONFH早期诊疗提供一种新思路与干预靶点。