基因工程技术制备自组装LL-37抗菌肽纳米胶束对脓毒症诱导的急性肺损伤的作用

    Effect of Self-assembled Antibacterial Peptide LL-37 Nanomicelles Prepared by Genetic Engineering Technology on Sepsis Induced Acute Lung Injury

    • 摘要:
      目的  通过基因工程技术制备自组装的LL-37抗菌肽纳米胶束,并评估这种新型多肽类药物在脓毒症诱导的急性肺损伤中的治疗潜力。
      方法 利用基因工程技术构建LL-37-ELPs融合蛋白表达载体,利用ELPs 融合蛋白的可逆相变特性,经过多次可逆相变循环从原核表达的可溶性蛋白上清溶液中特异性分离出含有ELPs标签的融合蛋白。透射电镜和动态光散射鉴定LL-37抗菌肽纳米胶束表征。随后构建脓毒症小鼠模型,并在术后5 d内,每天静脉注射药物进行治疗。统计5 d内的存活率。术后6 d,对各组小鼠安乐处死。HE染色法观察肺组织病理情况,并评估肺损伤得分。伊文思蓝染色评估肺内皮细胞通透性,并计算肺湿重比。ELISA检测肺组织炎性因子TNF-α、IL-1β和IL-6的表达情况。流式细胞术检测中性粒细胞聚集情况。
      结果 成功制备LL-37抗菌肽纳米胶束,大小均一,直径约为80~100 nm,稳定性好。在脓毒症小鼠中,能够有效减轻脓毒症诱导的肺损伤,减轻肺部炎症,提高脓毒症小鼠存活率。
      结论 成功制得LL-37抗菌肽纳米胶束,其在脓毒症诱导的急性肺损伤中具有良好的应用前景。

       

      Abstract:
      OBJECTIVE To prepare self-assembled antimicrobial peptide LL-37 nanomicelles by genetic engineering technology and evaluate the therapeutic potential of this novel peptide in sepsis induced acute lung injury.
      METHODS Using genetic engineering technology to construct LL-37-ELPs fusion protein expression vector, taking advantage of the reversible phase transition property of ELPs fusion protein, the fusion protein containing ELPs tag was specifically isolated from the supernatant of soluble protein expressed in E. coli by several inverse transition cycle. Transmission electron microscopy and dynamic light scattering were used to characterize antimicrobial peptide LL-37 nanomicelles. Subsequently, a mouse model of sepsis was established, and drugs were injected intravenously every day for 5 d after surgery. Survival rates within 5 d were counted. Six days after surgery, mice in each group were sacrificed peacefully. HE staining was used to observe the pathological changes of lung tissue, and the lung injury score was evaluated. Lung endothelial cell permeability was assessed by Evans Blue staining, and lung wet weight ratio was calculated. ELISA was used to detect the expression of tumor necrosis factor-α, IL-1β and IL-6 in lung tissue. Neutrophil aggregation was detected by flow cytometry.
      RESULTS The LL-37 antimicrobial peptide nanomicelles were successfully prepared, which were uniform in size with a diameter of 80−100 nm and good stability. In septic mice, it could effectively reduce sepsis-induced lung injury, reduce lung inflammation, and improve the survival rate of septic mice.
      CONCLUSION Antimicrobial peptide LL-37 nanomicelles are successfully prepared, which have good application prospects in sepsis-induced acute lung injury.

       

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