气管内滴入核仁素shRNA表达载体对脂多糖肺损伤大鼠肺泡巨噬细胞活化的影响  

Influence of intratracheal instillation of pBS-U6. C23shRNA vector on the activation of alveolar macrophages in rats with lipopolysaccharide-induced acute lung injury

作　　者：王艺[1,2] 徐剑铖[1] 毛梅[3] 李秋梅 田静 苏磊 

机构地区：[1]第三军医大学新桥医院全军呼吸内科研究所,400037 [2]山东省济南市第六人民医院呼吸内科,重庆250200 [3]解放军324医院呼吸科,重庆400020

出　　处：《中华肺部疾病杂志（电子版）》2014年第2期16-20,共5页Chinese Journal of Lung Diseases(Electronic Edition)

基　　金：国家自然科学基金资助项目(30370631)

摘　　要：目的建立气管内滴入核仁素shRNA表达载体的实验模型,观察其对脂多糖(LPS)诱导急性肺损伤(ALI)大鼠肺泡巨噬细胞(AM)活化的影响,探讨AM膜核仁素在LPS致大鼠ALI中的作用及机制。方法选择健康雄性SD大鼠40只,随机分为A组(空白对照组),B组(ALI组),C组(转染重组质粒pBS-U6.C23shRNA并ALI组),D组(转染对照质粒pBS-U6.ControlshRNA并ALI组)。大鼠经尾静脉注射LPS以建立ALI模型,气管内滴入法转染重组质粒pBS-U6.C23shRNA表达载体,免疫印迹(Western blot)检测AM膜核仁素蛋白表达;核酸电泳迁移率实验(EMSA)检测AM中核因子(NF-κB)活性;酶联免疫吸附法(ELISA)检测肺泡灌洗液(BALF)中肿瘤坏死因子-α(TNF-α)和白介素-6(IL-6)表达。结果转染pBS-U6.C23shRNA表达载体后可以显著下调AM膜核仁素蛋白表达;减轻肺病理损伤;降低NF-κB的活性以及肺泡灌洗液中TNF-α和IL-6的含量。与A组相比,B组及D组的大鼠AM膜核仁素蛋白表达显著增加,组间有统计学差异(P<0.01);C组大鼠AM膜核仁素蛋白表达明显下调,与B组相比,组间差异有统计学意义(P<0.01);而D组与B组中的大鼠AM膜核仁素蛋白表达组间没有统计学差异(P>0.05)。经LPS处理后,各组大鼠AM中NF-κB活性显著增加,与A组相比,组间差异非常明显(P<0.01)。C组大鼠AM中NF-κB活性明显降低,与B组比较,差异显著(P<0.05);而D组与B组比较,组间无明显差异(P>0.05)。经LPS诱导肺损伤后,显著增加了大鼠BALF中促炎因子TNF-α和IL-6的表达,各组和A组相比差异显著(P<0.01);C组大鼠BALF中TNF-α和IL-6的含量显著降低,与B组相比差异显著(P<0.01);而D组与B组相比,组间差异无统计学意义(P>0.05)。结论气管内滴入法可以安全有效转染核仁素shRNA表达载体,抑制AM活化,减轻肺损伤,AM膜核仁素参与了LPS致大鼠ALI的发病过程。Objective To observe the efffection of cell-surface nucleolin in the activation of alveolar macrophages （AM） in rats with lipopolysacharide（LPS）-induced acute lung injury（ ALI）, and to investigate its potential role in the pathogenesis of ALI, we develop the experimental method of transfected pBS-U6-C23.shRNA vector by intratracheal instillation. Methods Healthy male SD rats were randomly divided into group A （blank control group） and group B （ALI）, group C （transfection recombinant plasmid pBS-U6. C23shRNA and ALI group）, group D （transfection controlled plasmid pBS-U6. ControlshRNA and ALI group）. The rat model of ALI was established by intravenous injection of LPS, the pBS-U6- C23. shRNA vector was transfected intratracally. Western blot was employed to analyze the expression of cell-surface nucleolin protein in AM. The activity of NF-kB in AM was detected by electrophoretic mobility shift assay （EMSA）. The concentrations of TNF-α and IL-6 in the bronchoalveolar lavage fluid （BALF） were assayed by enzyme linked immunosorbent assay （ELISA）. Results After transfected with pBS-U6. C23shRNA vector, the expression of cell-surface nucleolin protein, the histopathologic, the NF-κB activity and the levels of tumor necrosis factor-α（TNF-α） and interleukin-6 （IL-6） decreased remarkably. Compared with group A, the expression of cell-surface nucleolin protein of AM were significantly in group B and group D, the difference was statistically significant between groups （P 〈 0.01 ） ; it was significantly lower in group C , compared with group B, the difference was statistically significant between groups （P 〈 0.01 ） ; but there was no statistical difference between group B and group D （P 〉 0.05 ）. compared with group A, the NF-κB activity were increased significantly in group D and B, very obvious difference between groups （ P 〈 0.01 ）. compared with group B, the NF-κB activity was significantly decreased, significant difference between groups （ P

关 键 词：急性肺损伤 核仁素 脂多糖 肺泡巨噬细胞 肿瘤坏死因子-α 白介素-6 

分 类 号：R392[医药卫生—免疫学] R563[医药卫生—基础医学]