IL-23通过破骨细胞间接诱导强直性脊柱炎新骨形成的机制

Pathological new bone formation is the fundamental cause of disability in ankylosing spondylitis (AS), but the underlying mechanism is still unknown and there is no effective drug to prevent it. Physiologically, bone resorption and bone formation are coupled precisely. It is well known that a lot of factors indirectly regulate the differentiation and function of osteoclasts through osteoblasts. However, it is still unknown which factors indirectly regulate osteoblast differentiation through osteoclasts. Here, we are planning to study the possible effects of IL-23, which plays a critical role in the pathogenesis of AS, on bone formation indirectly modulated by osteoclasts. It will be studied in vitro whether IL-23 upregulated the expression of BMP、Wnt、S1P、HtrA1 on osteoclasts, whether the culture medium collected from IL-23-stimulated osteoclasts stimulates the proliferation of osteoblasts, the expression of osteoblast-differentiation related molecules, the production of bone matrix and bone mineralization, by using quantitative PCR, Western blotting, flow cytometry and specific staining. Furthermore, in vivo studies will be performed to elucidate whether the blocking of osteoclast differentiation by anti-RANKL neutralizing antibodies or osteoclast deficiency induced by c-fos knock-out inhibits new bone formation in mice with IL-23 overexpression, an animal model of AS, evaluated by histology and micro-CT. Taken together, different studies will be performed to clarify whether IL-23 indirectly induces new bone formation in AS.

病理性新骨形成是强直性脊柱炎(AS)致残的根本原因，但其发生机制不明，也无有效治疗药物。正常骨吸收和骨形成精确耦联。已发现多种因子通过成骨细胞间接调控破骨细胞的分化与功能，但对于通过破骨细胞间接调控成骨细胞分化的机制缺乏研究。我们拟研究在AS发病中起关键作用的IL-23是否通过破骨细胞间接促进骨形成。采用定量PCR、蛋白印迹、流式细胞术、特殊染色研究，IL-23能否调节破骨细胞表达BMP、Wnt、S1P、HtrA1等影响成骨细胞分化的分子；IL-23刺激的破骨细胞的培养上清能否调节成骨细胞的增殖、与分化相关的分子表达、骨基质的合成与骨矿化；阻断潜在中介分子能否抑制IL-23对成骨细胞的间接效应。采用组织学检查、微CT评估，抗RANKL抗体阻断破骨细胞的分化或敲除c-fos基因阻断破骨细胞发育能否抑制AS动物模型IL-23高表达鼠的新骨形成。从多个层面探索IL-23能否间接促进AS新骨形成。

强直性脊柱炎(ankylosing spondylitis, AS)是一种以慢性腰背痛为主要症状、并发脊柱强直等残疾的风湿病，肌腱端炎(enthesitis)是AS的基本病理改变，局部出现骨侵蚀，骨质破坏处反应性产生新骨，骨赘(新骨)形成是AS结构破坏和功能受损的决定性因素。本项目旨在探索AS并发新骨形成的机制，重点研究IL-23的作用机制。. 本课题首先在体外利用小鼠骨髓来源的单个核细胞成功诱导破骨细胞的分化与成熟；采用基因芯片研究了IL-23对破骨细胞基因表达谱的调节作用，结果显示IL-23可以显著调节破骨细胞内55 种基因的表达水平(上调23种、下调32种)；IL-23促进破骨细胞表达Ephrin B2、SPHK1等成骨分子，对semaphorin 4D及HtrA1等抑制成骨的分子无促进作用；利用大鼠颅骨骨髓来源的成骨细胞前体细胞在体外成功诱导成骨分化模型；检测了不同来源的成骨细胞表达IL-23、IL-17和IL-22的受体情况；研究了IL-23、IL-17和IL-22对成骨细胞增殖和分化的调节作用；小鼠尾静脉注射微环DNA建立了IL-23过表达诱导关节炎/脊柱炎的模型；敲低SOCS3可加重IL-23过表达鼠的关节炎症和骨赘程度。. 结论，IL-23可以促进破骨细胞表达促进成骨的分子而不促进抑制成骨的分子；大鼠骨髓来源的成骨细胞前体可以高表达IL-17受体、低表达IL-22 和IL-23受体；三种成骨细胞株MC3T3-E1、C2C12、Saos-2细胞均不能完整表达IL-23、IL-22或IL-17的各受体亚单位；IL-17轻度抑制成骨细胞分化，IL-23和IL-22均不能调节成骨细胞的增殖或分化；IL-23过表达可诱导脊柱关节炎模型，敲低SOCS3可加重该模型。