SDF-1/CXCR4异构复合体在关节软骨炎症损伤/干细胞修复功能平衡中的作用

Mesenchymal stem cell (MSC) can be regulated and directed to differentiate into steady-state precartilaginous stem cells (PCSC). The MSC were anchored in specific nest, whose de-adhesion-homing-colonization is the critical factor to activate the reparation of the damaged cartilage. Stromalcell-derivedfactor-1(SDF-1)/ CXCchemokinereceptor4(CXCR4) axis is the main induced signal for MSC homing to repair the damaged cells. Meanwhile, this axis can also trigger the RC inflammatory injury. The mechanism about the regulation of the two contradictory functions and how MSC can firmly colonize to the target region is still unclear. We speculate that: (1) the phosphorylation state of the active groups between inflammatory SDF-1 and chemotactic SDF-1 is different. They can form different active groups complex with CXCR4; ②The downstream activation pathways of the two complex is different: the former can lead to the cartilage damage through the G protein-dependent signaling pathway；however, the latter can induce the cartilage reparation through the non-G-protein-dependent signaling pathway；(3) the latter also activates of MSC superficial specific affinity and adhesion molecule to realize the colonization purpose. To proof the assumptions, the project intends to adopt proteomics method to detect inflammation、secondary structure and tertiary structure of chemotactic SDF-1 protein, and phosphorylation state of active groups; to detect subsequent activation pathway of each active S/C complex; and finally to detect the active effect of this axis to MSC adhesion molecule. The research may validate the mechanism that the S/C axis can induce MSC colonization. The research will provide theoretical basis for precise regulation of MSC differentiation and promotion reconstruction of cartilage.

SDF-1/CXCR4轴既能促MSCs动员、定向分化，进而修复损伤关节软骨；又能激活细胞外信号调节酶(Erk)及p38MAPK通路造成软骨损伤，这两个相反的功能如何平衡？ SDF-1具有相反作用的异构体α和β；而SDF/CXCR4复合体下游可激活G蛋白依赖转导通路和非G蛋白依赖通路。故推测：1.SDF-1的α和β异构体分别表现出软骨炎症损伤及修复作用，该区别由于活性基团的磷酸化状态不同；2.α异构体通过G蛋白依赖通路发挥软骨炎症损伤效应；β异构体通过非G蛋白依赖通路发挥干细胞修复效应。 为验证假设,1.我们采用蛋白质组学检测方法，检测SDF-1两个异构体的蛋白质二级和三级构象以及磷酸化活性基团的不同，研究α和β异构体的功能；2.分别研究α和β异构体的炎症性和趋化性的表达机制及其对MSC的趋化诱导作用；3.S/C轴调控MSCs在受损软骨部位定植的机制。为精确调控MSC分化,促软骨重建提供依据

项目背景：关节软骨缺损是临床上常见的病症，骨髓间充质干细胞（mesenchymalstem cells，MSCs）是一种理想的多能干细胞，定向分化为稳定状态的 PCSCs而修复软骨损伤。SDF-1/CXCR4 轴既能促 MSCs 动员、定向分化，进而修复损伤关节软骨；又能激活细胞外信号调节酶(Erk)及 p38MAPK 通路造成软骨损伤。SDF-1 具有相反作用的异构体α和β；而 SDF/CXCR4 复合体下游可激活 G 蛋白依赖转导通路和非 G 蛋白依赖通路。.主要研究内容：1) SDF-1对体外骨髓间充质干细胞(MSCs)和软骨细胞软骨分化和成熟的影响 ；2) SDF-1α/CXCR4 复合体与 SDF-1β/CXCR4 复合体功能检测；3) SDF-1/CXCR4 两种异构复合体的下游信号途径检测；4) SDF-1/CXCR4 轴调控MSCs 在受损软骨部位定植的机制。.重要结果：1.SDF-1 的α和β异构体分别表现出软骨炎症损伤及修复作用，该区别由于活性基团的磷酸化状态不同。2.α异构体通过 G 蛋白依赖通路发挥软骨炎症损伤效应；β异构体通过非 G 蛋白依赖通路发挥干细胞修复效应。3. SDF-1α在软骨分化诱导培养条件下促进MSCs软骨成熟蛋白CollagenX和MMP-13的表达，但对软骨分化早期指标Collagen II和Aggrecan 的表达以及软骨基质形成没有影响。4. SDF-1α可以促进MSCs 中Wnt/β-catenin通路的激活；抑制Wnt/β-catenin之后，发现SDF-1α对MSCs软骨分化作用消失。5. SDF-1α促进软骨损伤的另一机制在于通过激活MAPK途径（包括ERK和p38，但不包含JNK）促进破骨细胞的形成，并且促进抗酒石酸酸性磷酸酶TRAP，组织蛋白酶K（CK）和基质金属蛋白酶（MMP）-9在破骨细胞中的表达，从而加速软骨下骨的吸收。6. SDF-1β/CXCR4 复合体通过降低ADAMTS-5的表达和蛋白聚糖的丢失来保护骨关节炎软骨。.科学意义：通过分别研究α和β异构体的炎症性和趋化性的表达机制及其对 MSC 的趋化诱导作用以及S/C 轴调控 MSCs 在受损软骨部位定植的机制，为精确调控 MSC 分化,促软骨重建提供依据。