透明质酸修饰的壳聚糖CrmA/IL-1Ra纳米粒子对骨关节炎软骨的保护作用研究

Cytokine response modifier A (CrmA) and IL-1 receptor antagonist (IL-1Ra) can block the formation and function of IL-1β which plays a pivotal role in osteoarthritis (OA). Chitosan (CS) modified by hyaluronic acid (HA) can protect chondrocytes by itself and be loaded with object proteins or target genes as a vector to treat OA. Our previous study has showed that HA-CS-CrmA/IL-1Ra microspheres can inhibit inflammation and apoptosis in chondrocytes. However, the release duration of proteins from the microspheres is too short. HA-CS can construct nanoparticles with CrmA/IL-1Ra through electrostatic interaction and then bind with CD44 receptor existing on the surface of chondrocytes and synoviocytes to make them continuously express the object proteins, which finally performs targeted gene therapy. We plan to build HA-CS-CrmA/IL-1Ra nanoparticles to transfect synoviocytes and co-culture with cartilage, so as to make the transfected synoviocytes express CrmA and IL-1Ra which can interrupt the secretion and function of IL-1β and broadly protect chondrocytes. These results will provide theoretical and experimental evidence for the treatment of OA.

细胞因子反应调节蛋白A（CrmA）和白介素1受体拮抗剂（IL-1Ra）可以明显阻止骨关节炎（OA）主要致病因子IL-1β的合成及功能。透明质酸（HA）及壳聚糖（CS）本身对关节软骨有保护作用，又可作为生物载体装载目的蛋白和基因治疗OA。我们的前期研究已经证明HA-CS-CrmA/IL-1Ra缓释微球能明显抑制IL-1β诱导的软骨细胞炎症及凋亡，但其蛋白缓释过程持续时间较短。HA-CS可以通过静电作用和CrmA/IL-1Ra质粒聚合形成纳米粒子，能与软骨细胞和滑膜细胞膜表面CD44受体特异结合，使其持续表达目的蛋白，进而实现靶向基因治疗的目的。本项目拟构建HA-CS-CrmA/IL-1Ra纳米粒子，转染滑膜细胞，使其有效表达CrmA和IL-1Ra目的蛋白，与OA软骨组织共培养，阻断IL-1β的分泌及其下游炎症反应，多方位保护关节软骨，为临床更为有效治疗OA提供理论和实验依据。

细胞因子反应调节蛋白A（CrmA）和白介素1受体拮抗剂（IL-1Ra）可以明显阻止骨关节炎（OA）主要致病因子IL-1β的合成及功能。透明质酸（HA）及壳聚糖（CS）本身对关节软骨有保护作用，又可作为生物载体装载目的蛋白和基因治疗OA。HA-CS可以通过静电作用和CrmA/IL-1Ra质粒聚合形成纳米粒子，能与软骨细胞和滑膜细胞膜表面CD44受体特异结合，使其持续表达目的蛋白，进而实现靶向基因治疗的目的。结果显示CS/HA纳米粒子具有合适的粒径和电势；凝胶阻滞测定显示质粒DNA可以被有效保护并持续释放；细胞毒性结果显示CS/HA/pDNA纳米粒子可以安全作用于滑膜细胞；RT-PCR和western blot表明CS/HA/pDNA纳米粒子能够增加原代滑膜细胞IL-1Ra及CrmA的表达，降低IL-1β诱导的炎性因子在滑膜细胞中的表达；进一步，证实该纳米复合物在大鼠OA模型中的软骨保护作用。我们的研究结果表明CS/HA/pDNA纳米粒子具有骨关节炎治疗的潜在价值。