载异烟肼及利福平白蛋白纳米粒治疗兔脊柱结核的实验观察

Experimental study of the treatment of spinal tuberculosis in rabbit with isoniazid and rifampicin loaded albumin nanoparticles in rabbits

中文关键词:  白蛋白  纳米粒  异烟肼  利福平  脊柱结核

英文关键词:Albumin  Nanoparticles  Isoniazid  Rifampicin  Spinal tuberculosis

中文摘要:

  【摘要】 目的：观察载异烟肼（isoniazid，INH）、利福平（rifampicin，RFP）白蛋白纳米粒治疗兔脊柱结核的临床疗效。方法：以结核分枝杆菌H37Rv标准株构建新西兰大白兔脊柱结核模型，选取造模成功的新西兰大白兔35只。随机分为3组，实验组（A组）和对照组（B组）各14只，空白对照组（C组）7只。A组耳缘静脉滴注载INH、RFP白蛋白纳米粒（INH 3mg/kg，RFP 4mg/kg，一周一次，共12周）。B组耳缘静脉滴注INH和RFP普通注射液（INH 3mg/kg，RFP 4mg/kg，一天一次，共12周）。C组耳缘静脉滴注生理盐水（7mg/kg，一天一次，共12周）。观察三组给药后6周、12周各组脊柱结核兔的体重及病灶CT变化，并在给药的6周和12周每组各处死3只兔留取病灶椎体组织标本病理检查，采用高效液相色谱法测定血液、病灶椎体、正常椎体及肌肉组织中两种药物浓度，评估载INH、RFP白蛋白纳米粒对于兔脊柱结核模型的临床疗效。结果：A组14只模型兔经治疗后全部成活且体重均增加，治疗6周、12周后纳米粒组兔平均体重为2.94±0.11kg、3.04±0.18kg，与治疗前比较差异有统计学差异（P<0.05）。B组14例模型兔经治疗后均存活，4例出现后腿瘫痪的神经症状。治疗6周、12周后平均体重维持在2.5±0.2kg，与治疗前相比无统计学差异（P＞0.05）。A组治疗6周时组织病理观察到椎体呈现骨质破坏的炎症修复背景，大量淋巴细胞和嗜酸性粒细胞，还有一些浆细胞存在，治疗12周时后组织病理可见大量的成骨细胞和已经修复完成的骨质结构骨小梁形成，影像学显示模型病灶处碎片骨组织消失，周围脓肿和坏死被正常骨组织替代，病椎基本治愈。B组治疗6周时病灶椎体组织病理提示椎体骨质破坏炎症反应重，大量的坏死物及纤维素性渗出物。CT扫描提示病椎有部分修复，但仍有碎片征。治疗12周时病灶椎体组织病理可见椎体大量破骨细胞、间充质细胞、毛细血管，显示病灶处于修复期。CT显示病椎为不全修复，碎片征基本消失，但椎体内仍然存在小的空洞，周边硬化带形成。治疗6周时，A、B两组INH、RFP血浆药物浓度差异有统计学意义（P<0.05），病灶椎体INH、RFP有效骨组织药物浓度差异有统计学意义（P<0.05），肌肉组织INH、RFP药物浓度差异无统计学意义（P<0.05），治疗12周时两组INH、RFP血浆药物浓度差异有统计学意义（P<0.05），病灶椎体INH、RFP有效骨组织药物浓度较差异有统计学意义（P<0.05），肌肉组织INH、RFP有效组织药物浓度差异无统计学意义（P＞0.05）。C组分别在给予安慰剂生理盐水耳缘静脉滴注45~60d内死亡。结论：与INH、RFP普通制剂相比，载INH、RFP白蛋白纳米粒具有良好的体内缓释特性以及病灶骨组织中的有效生物分布，在兔脊柱结核模型中的治疗效果显著优于普通制剂。

英文摘要:

  【Abstract】 Objectives: To observe the clinical efficacy of isoniazid(INH) and rifampicin(RFP) loaded albumin nanoparticles in the treatment of spinal tuberculosis in rabbits. Methods: New Zealand white rabbits with the standard strain of Mycobacterium tuberculosis H37Rv were selected to establish the spinal tuberculosis model. Thirty-five New Zealand White Rabbits were divided into 3 groups: experimental group(14 rabbits), control group(14 rabbits) and blank control group(7 rabbits). INH and RFP loaded albumin nanoparticles were administrated forthe rabbits of group A(INH 3mg/kg, RFP 4mg/kg, once per week for a total of 12 weeks). Ordinary INH and RFP were administrated for the rabbits of group B(INH 3mg/kg, RFP 4mg/kg, once per day for a total of 12 weeks). Normal saline was administrated for the rabbits of blank control group(7mg/kg, once per day for a total of 12 weeks). The body weight was measured and CT scan was performed for all the rabbits in the 3 groups at 6 weeks and 12 weeks after treatment started, and 3 rabbits in each group were sacrificed. The concentrations of INH and RFP in blood, infected vertebral body, normal vertebral body and muscle tissue were tested by high performance liquid chromatography(HPLC). The efficacy of INH and RFP loaded albumin nanoparticles in the treatment of spinal tuberculosis in rabbit model was evaluated. Results: For group A, after 6 weeks of treatment, the weight of the rabbits increased by 0.44±0.09kg, and 0.27±0.07kg after 12 weeks of treatment. After 6 weeks of treatment, the pathological result showed inflammatory repair after bone destruction in the backgroud, together with a large number of lymphocytes, eosinophils and some plasma cellsaccumulating. After 12 weeks of treatment, a large number of osteoblasts and trabecular bone formation were observed in group A. CT showed bone fragments disappeared and abscess and necrosis were replaced by normal bone tissue. In group B, all the 14 rabbits survived and 4 had neurological symptoms of hind leg paralysis. There was no significant increase in body weight after 6 weeks of treatment, the average body weight was maintained at 2.5±0.2kg. Only 6 rabbits had increased weight of an average of 0.30±0.07kg after 12 weeks of treatment. At 6 weeks, vertebral bone destruction and severe inflammation were observed as well as massive necrosis and fibrin formation. CT scan revealed partial repair of the infected vertebrae, but there were still debris signs. After 12 weeks of treatment, a lot of osteoclasts, mesenchymal cells and capillaries were found at the infected vertebrae, indicating the repair of lesion. CT showed incomplete repair of the lesion represented by disappearance of bone fragments and cavities formation surrounded by sclerosis band. 6 weeks of treatment, there was a significant difference in plasma concentration of INH and RFP between two groups(P<0.05). The concentrations of INH and RFP were measured at the infected vertebraewhich were significantly different from those of group B. Muscle tissue INH, RFP measured of the tissue concentration were no significant difference compared with the group B. 12 weeks of treatment, there was a significant difference in plasma concentration of INH and RFP between two groups(P<0.05). The concentrations of INH and RFP were measured at the infected vertebraewhich were significantly different from those of group B. Muscle tissue INH, RFP measured of the tissue concentration were no significant difference compared with the group B. Rabbits in group C died within 45-60 days of the placebo normal saline administration. Conclusions: Compared with control group, the INH-RFP loaded albumin nanoparticles have good sustained release characteristics in vivo and effective biodistribution in the infected bone tissue. Its rapeutic effect in spinal tuberculosis in rabbit model is significantly better than that of conventional preparation.