靶向调控SDF-1/CXCR4信号通路干预关节软骨退变的分子机理研究

As the population ages,the prevalence rate of osteoarthritis (OA)distinguished by cartilage degeneration increases daily, and targeting for SDF-1/CXCR4 signaling pathway could regulate articular cartilage degeneration, so in this study,the high effective and biological stable T140 analog TN14003 is used to block SDF-1 / CXCR4 signaling pathway,T140 and AMD3100 are used as control study.The OA cartilage degeneration will be regulated through molecular,cellular, tissue level and in vivo.The molecular mechanism of the antagonists is investigated with photoaffinity labeling, and the biological effect of OA chondrocytes is observed after blocking SDF-1 / CXCR4 signaling pathway. The histological observation, ELISA assay, RT-PCR and Western blot are used to observe the cartilage tissue degradation of OA patients in vitro. On this basis,TN14003,T140 and AMD3100 are administered with Alzet pump in Hartley guinea pig animal model with primary OA to block SDF-1/CXCR4 signaling pathway and explore the regulation the degeneration of OA cartilage tissue in vivo.The both knee cartilage tissue are collected and observed with histological observation, immunohistochemical staining, ELISA assay, RT-PCR and Western blot.The aim is to explore the new high effective medicine targeting for SDF-1 / CXCR4 signaling pathway and prevent the degenerative pathological process of OA cartilage, it could provide the experimental basis and explore the clinical high effective and safe targeting drugs for OA.

随着社会人口的老龄化，以关节软骨退变为特征的OA患病率日益剧增，而靶向SDF-1/CXCR4信号通路可调控关节软骨退变，本研究用高效稳定的拮抗剂TN14003阻断SDF-1/CXCR4信号通路，并与T140及AMD3100对照，从分子、细胞、组织及体内水平调控OA软骨退变，用光亲和标记技术探讨各拮抗剂的分子作用机理，观察其阻断SDF-1/CXCR4信号通路对OA软骨细胞的生物学效应，经组织学观察、ELISA测定、RT-PCR和Western blot检测OA患者软骨组织体外降解情况。在此基础上，用Alzet微量泵于原发性OA豚鼠体内予靶向药物TN14003、T140及AMD3100，探讨其体内调控OA软骨退变的作用， 经组织学观察，免疫组化染色，ELISA测定，RT-PCR及Western blot检测，探寻阻止OA软骨病理进程新的靶向药物，为研发高效安全的OA靶向药物提供实验依据。

随着社会人口的老龄化，OA患病率日益剧增，而SDF-1/CXCR4信号通路能介导膝关节OA发生发展，且靶向阻断SDF-1/CXCR4信号通路可有效延缓关节软骨退变。本研究将TN14003、T140及AMD3100加入到人OA软骨细胞中培养后发现三种拮抗剂给药剂量下对OA软骨细胞增长及增殖无影响，且通过减少MMP-3、MMP-13分泌并降低Ⅱ型胶原蛋白和聚集蛋白聚糖降解调控人OA软骨细胞退变，其中以TN14003效果最佳。但培养基中MMP-9未被检测到，可能由于培养基中MMP-9含量极低；采用计算机模拟技术模拟TN14003、T140及AMD3100分别与CXCR4的结合模式,明确三种拮抗剂与CXCR4结合强弱依次是：TN14003>T140>AMD3100，并经光亲和标记探针获知TN14003、T140、AMD3100小分子药物作用部位为细胞膜，且光亲和标记探针制备后对SDF-1诱导的趋化效应没有明显差异；将80块OA患者软骨组织块体外培养后加入TN14003、T140及AMD3100，发现三种拮抗剂皆能明显延缓OA软骨形态学退变，减少培养液内MMP-3、9、13分泌以及Ⅱ型胶原蛋白和聚集蛋白聚糖降解，且TN14003最佳，T140次之，AMD3100最差；在体外研究基础上，用Alzet微量泵于96只原发性OA Duncan-Hartley豚鼠体内予靶向药物TN14003、T140及AMD3100，12周后处死并收集双膝关节软骨行组织学检测及改良Mankin评分发现三种拮抗剂皆可延缓豚鼠OA模型的膝关节软骨组织退变，其中以TN14003最强，T140次之，AMD3100最差；免疫组化染色显示三种拮抗剂体内使IL-1，TNF-α的表达量下降，其中TN14003效果最佳；分子生物学检测发现三种拮抗剂皆可减少软骨细胞分泌和释放MMP-3、MMP-9、MMP-13，减少II型胶原蛋白，聚集蛋白聚糖的降解，可增加II型胶原蛋白，聚集蛋白聚糖合成，达到延缓OA软骨关节软骨的退变，其中以TN14003效果最佳，T140次之，AMD3100最差，其阻断效果差异是由于三种拮抗剂结合CXCR4的能力不同所致；因TN14003具有更好的血清稳定性和更低的细胞毒性，有望开发成为OA的靶向治疗药物，为今后进一步探索高效且副作用小的CXCR4靶向药物提供新的实验依据。