Mac-1通过上调Syk表达促进RANKL诱导破骨细胞分化的机制研究

Osteoclasts are cells of hematopoietic origin that decalcify and degrade the bone matrix, which are large, multinucleated cells formed by the fusion of precursor cells of monocyte/macrophage lineage. The differentiation and activation of osteoclasts are key points to wear debris particles induced peri-implant osteolysis. Mac-1 is an important integrin which mainly expresses on the surface of leukocytes and consisted with two subunits (CD11b/CD18). It involves in cells interaction and migration in many kinds of inflammatory diseases. Recently, Mac-1 is shown to be of great importance in the process of mononuclear pre-osteoclast cells fusion. However, the relationship between Mac-1 and osteoclast differentiation remains to be elucidated. We found the up-regulated expression of Mac-1 induced by TNF-α in our previous work. When CD11b blocking antibody applied, the RANKL induced osteoclast formation and UHMWPE (Ultra High Molecular Weight Polyethylene) particles induced bone resorption were inhibited and the expression of phosphorylated Syk protein decrease with a same trend. Therefore, we assume that Mac-1 can promote the differentiation of osteoclast with synergies of RANKL by up-regulating the Syk pathway. In this study, we are trying to clarify the molecular mechanism of Mac-1 in promoting osteoclast differentiation and activation through both in vivo and in vitro ways. This might provide a new potential therapeutic target for treating wear debris particles induced peri-prostheses osteolysis.

破骨细胞分化和激活是人工关节置换术后假体周围骨溶解发生的重要基础。通过本课题组前期研究发现，破骨细胞分化受其前体细胞表达的巨噬细胞分化抗原1（Macrophage antigen-1,Mac-1）调控，但具体机制并不清楚。我们的研究显示，Mac-1可以显著促进RANKL作用下的破骨细胞分化、成熟；阻断Mac-1则能显著减少破骨细胞的形成并抑制磨损颗粒诱导的骨溶解发生；此作用是通过破骨细胞前体细胞内脾酪氨酸激酶（Syk）信号实现的。因此，我们认为Mac-1在RANKL诱导的破骨细胞分化和磨损颗粒诱导的骨溶解中起协同作用，Syk通路是其主要靶点。本项目拟通过体内、体外研究明确Mac-1协同RANKL作用时破骨细胞前体细胞中Syk通路上相关分子表达情况的变化，同时研究Mac-1与RANKL通路中重要调控因子及其与破骨细胞分化标志分子之间的关系，明确Mac-1在促进破骨细胞分化中作用的分子机制。

由于人工关节置换术后假体周围骨溶解导致的假体无菌性松动已经成为人工关节翻修手术的主要原因，且这给患者的日常生活带来极大不便，而再次手术翻修也增加了患者的痛苦、经济负担、消耗了社会医疗资源。破骨细胞是这一病理过程中的核心效应细胞，植入人工假体磨损产生的颗粒诱导的破骨细胞分化、激活是这一病理过程的核心组成部分。因此，以破骨细胞为靶点的抗骨溶解研究对于预防人工关节无菌性松动具有十分重要的临床意义。本课题研究着眼于探究Mac-1分子在无菌性松动患者关节周围滑膜组织中的表达特点，探究Mac-1分子在RANKL诱导的破骨细胞分化中的作用及分子机制，同时分析TNF-α对Mac-1分子表达的影响。实验通过免疫组化染色、抗体封闭、基因沉默、TRAP染色、real-time PCR、western-blot、显微CT和三点弯曲实验等技术手段发现：CD11b表达阳性的患者其关节置换术后假体无菌性松动的发生率均高于CD11b表达阴性的患者（P<0.01），该分子可通过募集下游Syk，激活Syk通路，促进Erk的磷酸化水平，促进c-Fos的表达，最终通过上调破骨细胞核心转录因子NFATc1的转录，促进RANKL诱导的破骨细胞分化；而CD18亚基并不具备相同作用。CD11b缺失可抑制颗粒诱导的小鼠颅骨骨溶解，并导致小鼠股骨骨密度及刚度的升高。TNF-α可促进破骨前体细胞表面CD11b的表达，进而促进破骨细胞的分化。Mac-1分子过表达与人工髋关节置换术后假体无菌性松动的发生具有明确相关性，Mac-1分子的CD11b亚基可通过激活Syk通路促进RANKL诱导的破骨细胞分化，TNF-α可促进破骨前体细胞表面CD11b的表达，进而促进破骨细胞分化。该研究结论为粘附分子在破骨细胞分化激活过程中发挥作用的相关研究奠定基础，为Mac-1细胞膜表面分子所致破骨细胞前体细胞分化的信号通路研究提出新的可能，为从机制上预防人工关节置换术后假体周围骨溶解验证了新的途径。