复合TNF-a siRNA INH RFP纳米骨局部治疗脊柱结核的实验研究

Tumor necrosis factor-α plays an important role in inducing inflammation and osteoclasts' activation, which might accelerate osteolysis and lead to immunopathogenesis of granulumatous formation in the focus of tuberculosis. Our early study had demonstrated that anomaly of bone marrow-blood barrier caused by granulumatous formation and bone sclerosis prevents the biodistribution of first line antituberculosis drugs from normal vertebrae to focus and lead to the bad effects of antituberculosis therapy. Based on the studies of our pre grants of national natural science foundation ,the present study is going to develop a new nano- composite bone which combined with tumor necrosis factor-a(TNF-a) siRNA , namoparticles of isonicid and rifampin and hydroxyapatite ceramic, through the vitodynamics ,the release kinetics of in vitro and in vivo, the gene silencing effect and the therapeutic research of rabbit spinal tuberculosis, to explore the influences of TNF-a siRNA on bone marrow-blood barrier and the nano-medicine's targeting in pathologic vertebrae and its transport mechanism at marrow-blood barrier, meanwhile, the therapeutic effects of rabbit spinal tuberculosis is also observed. The study has great significance for the regional management of spinal tuberculosis, including the fast cure of focus and good reconstruction of pathologic vertebrae. It might provide a new thinking of improving effects, decreasing relapse of focus and deformity of diseased vertebral segment for the treatment of spinal tuberculosis.

结核病灶中的肿瘤坏死因子α(TNF-α)可诱发炎症和破骨细胞活化，加速骨溶解；并且诱导免疫病理反应,在结核核心病灶周围形成致密肉芽肿。我们的前期研究证实结核肉芽肿及其周边的椎骨硬化形成可导致病椎骨髓-血屏障异常，严重影响一线抗结核药物的组织穿透，致使疗效不佳。基于前期国家自然科学基金研究基础，本研究拟采用现代纳米技术和局部靶基因沉默生物技术，制备一种复合TNF-a siRNA 的INH RFP羟基磷灰石纳米骨，通过对复合材料的生物力学、体内外降解与释药、病灶局部基因沉默效应以及兔脊柱结核模型治疗的研究，探索TNF-αsiRNA对病椎病理性骨髓-血屏障形成机制的影响以及缓释纳米粒在病椎局部的靶向性和穿越骨髓-血屏障的转运机制，同时观察该复合纳米骨治疗兔脊柱结核的效果。此研究对于通过局部抗结核治疗实现快速治愈病灶和病椎结构性重建有重要意义，为临床治疗脊柱结核提供一种新的思路。

本研究采用现代纳米技术和局部靶基因沉默生物技术，成功构建了靶向TNF-α基因shRNA表达载体，结核菌素纯蛋白衍生物（PPD）可以诱导破骨细胞形成，且破骨细胞形成与PPD之间只有时间依赖性，无剂量依赖性；TNF-α在脊柱结核破骨细胞的形成中起着重要的作用，TNF-α基因shRNA能使TNF-α的表达下调，并抑制破骨细胞的形成。成功制备出分散均匀、大小均一的羟基磷灰石纳米颗粒。采用透射电子显微镜、X射线衍射分析、红外光谱分析对制备的载异烟肼、利福平纳米羟基磷灰石-硫酸钙人工骨进生物力学测试及体外释药研究。人工骨在体外降解稳定，并具有持续时间较长的且有效的缓释性能。在体内缓释性能较稳定，有效药物浓度可维持较长时间，初步实现了人工骨既能提供结构支撑又能缓释高效杀菌药的设计目标。通过生物相容性的体内外试验证实载异烟肼、利福平纳米羟基磷灰石-硫酸钙人工骨具有良好的生物安全性及生物相容性。采用高效液相色谱测定血液及组织分布浓度、从CT及三维重建、大体解剖、大体病理、骨组织HE/TRAP染色等手段，完成了复合纳米人工骨体内血浆及组织（病灶椎旁肌，病灶椎体）释药动力学研究、临床疗效观察。观察到复合材料体内缓慢降解及对结核模型骨缺损区良好的植骨界面修复和骨愈合情况。复合纳米人工骨作为植骨材料提供良好的结构性支撑和界面融合，修复和重建脊柱稳定性，防治了脊柱畸形。此研究对于通过局部抗结核治疗实现快速治愈病灶和病椎结构性重建有重要意义，为临床治疗脊柱结核提供一种新的思路。