TN14003壳聚糖温敏型凝胶制剂关节内靶向递药调控骨性关节炎软骨退变研究

With aging of population in the society, the prevalence rate of osteoarthritis(OA) is growing rapidly.Our previous studies showed that antagonists of CXCR4 could regulate degeneration of articular cartilage by blocking SDF-1/CXCR4 signaling pathway.The TN14003 has the characteristics of higher antagonistic efficiency,well stability and safety.So, we hypothesize that OA can be treated by delivering targeted TN14003 drug in joint if we develop new intelligent thermosensitive hydrogel with TN14003.It could not only improve the local targeted drug efficacy,but also avoid the first pass elimination of oral drug and whole body side effects. Therefore,this study was designed through optimizing TN14003 chitosan thermosensitive hydrogel with central composite design-response surface methodology,its physicochemical properties,biological characteristics,efficacy and molecular mechanism regulating the OA cartilage degeneration by targeted inteligent drug delivery with TN14003 chitosan thermosensitive hydrogel will be systemetic deeply studied at the level of molecule,cell,tissue and Diannan small ear pig OA model in vitro and vivo.The flow cytometry,gross observation,cartilage tissue histological observation and scores,immunohistochemical staining,ELISA detection, RT-PCR and Western blot detection will be used to explore new local targeted drugs for regulating OA cartilage degeneration in joint.This reseach will provide a new experimental basis on developing high efficacy,safety and local targeted drugs for OA treatment.

随着社会人口的老龄化，骨性关节炎（OA）患病率日益剧增，我们前期研究表明CXCR4拮抗剂可阻断SDF-1/CXCR4信号通路调控关节软骨退变，而TN14003拮抗效率高、稳定性好、安全性佳。为此提出假说：若将TN14003制成新型温敏智能凝胶关节内靶向递药治疗OA，既提高局部靶向药物疗效，又避免口服制剂首过消除效应及全身应用不良影响。故本研究采用星点-效应面法优化制备TN14003壳聚糖温敏型凝胶，从分子、细胞、组织及滇南小耳猪OA模型体内多层面系统深入研究TN14003壳聚糖温敏型凝胶的理化性质、生物学特性及关节内靶向递药阻断SDF-1/CXCR4信号通路调控OA软骨退变效果及分子机理，经细胞毒性、流式细胞分析、大体观察、软骨组织学观察评分、免疫组化染色、ELISA测定、RT-PCR及Western blot检测，探寻关节内靶向调控OA软骨退变新药，为研发高效安全的OA靶向药物提供依据。

随着社会人口的老龄化，骨性关节炎（OA）患病率日益剧增，项目组前期研究发现TN14003可与SDF-1竞争性拮抗CXCR4，延缓关节软骨退变，但TN14003用于动物体内的作用时间有限。故本项目在前期研究基础上找到壳聚糖和β甘油磷酸钠的合理配方并制备出短时间内可成胶的温度敏感型凝胶，该凝胶可负载TN14003，并对OA软骨细胞具有良好的生物相容性，不影响细胞增殖。将TN14003温敏型凝胶加入到OA软骨细胞中观察发现，TN14003温敏型凝胶可抑制软骨细胞分泌MMP-3、MMP-13，同时促进软骨细胞内ColⅡ及ACAN表达。本项目还建立了滇南小耳猪膝关节OA模型，将TN14003温敏型凝胶注射到动物膝关节腔内可长效并高效地延缓关节软骨退变，抑制滑膜分泌SDF-1和IL-1β，抑制软骨细胞分泌MMP-3、MMP-13，同时促进软骨细胞内ColⅡ及ACAN表达。本项目还基于基因芯片技术发现miR-146a-5p与TN14003具有协同作用，并能直接抑制靶基因CXCR4表达，进而抑制软骨细胞分泌MMP-3、MMP-13，同时促进软骨细胞内ColⅡ及ACAN表达。因此，本项目成功制备出具有缓释作用的TN14003温敏型凝胶，在体内及体外皆能高效延缓软骨退变；同时miR-146a-5p参与了该过程的调控，为制备高效安全的关节腔靶向递药系统奠定了扎实的研究基础。