摘要目的 探讨气道剪应力(SS)作用下黏蛋白(MUC)5 AC胞外极向分泌的主要介导分子.方法 常规培养16HBE细胞,使用Stata软件产生随机数字随机分为5组并分别进行如下处理:A组(对照组):10％胎牛血清(FBS)+DMEM高糖培养液继续培养,不再予任何刺激;B组(SS组):只给予节律性旋转装置产生的SS刺激;C组[SS+Rac-1特异性抑制剂(NSC23766)组]:给予SS刺激前30 min,加入NSC23766(10 μmol/L);D组(SS+微丝肌动蛋白解聚剂组):给予SS刺激前30 min,加入细胞松弛素D(100 μg/L);E组[SS+皮层肌动蛋白结合蛋白(Cortactin)小干扰RNA(siRNA)转染组]:用成功转染Cortactin-siRNA的细胞予以SS刺激.每个实验组设6个复孔,重复3次实验用于统计学分析.采用节律性旋转装置模拟呼吸时气流对气道产生的SS作用.用Cortactin-siRNA转染抑制Cortactin功能.酶联免疫吸附试验(ELISA)法测定细胞培养上清液中MUC5AC的胞外分泌量;Westem印迹法检测Cortactin及其磷酸化(p-Cortactin)水平和转染效果;激光共聚焦显微镜观察微丝肌动蛋白的聚合情况.结果 Cortactin-siRNA转染成功.A、B、C、D、E组MUC5AC相对含量分别为0.210 ±0.013、0.631±0.025、0.473±0.112、0.330±0.067、0.272±0.019,B组均显著高于其他各组(P=0.000、0.043、0.000、0.000);B组Cortactin相对表达水平为0.670±0.048,显著高于E组的0.132±0.014(P＜0.01),但与A、C、D组的0.641 ±0.016、0.622±0.012、0.653±0.027比较差异均无统计学意义(均P＞0.05);B组p-Cortactin相对表达水平为0.582±0.067,显著高于A、C、E组的0.131 ±0.011、0.393±0.045、0.170±0.016(P =0.000、0.021、0.000),而D组(0.511 ±0.029)与B组差异无统计学意义(P =0.246).结论 Rac-1、Cortactin和微丝肌动蛋白是气道SS作用下MUC5AC胞外极向分泌的主要介导分子.

abstractsObjective To explore the main mediated molecules of mucin (MUC) SAC extracellular secretion stimulated by airway shear stress (SS).Methods The 16 human bronchial epithelial (HBE) cells were cultured and randomized divided by Stata software into 5 groups:A.control group; B.SS stimulated group; C.SS stimulated & NSC23766 (a specific inhibitor of Rac-1)incubated group; D.SS stimulated & Cytochalasin D incubated group; E.Cortactin-siRNA (a small interfering RNA of Cortactin) transfected & SS stimulated group.Each group consisted of 6 parallel wells.Triplicate experiments were performed for statistical analysis.Rhythmic rotating device was used to simulate the breathing air flow mediated shear stress.The function of Cortactin was inhibited by Cortactin-siRNA.The relative content of MUC5AC in supematant was measured by enzyme linked immunosorbent assay (ELISA).The p-Cortactin (phosphorylation Cortactin) relative level,Cortactin relative level and the effect of transfection were measured with Western blotting.And laser confocal microscope was used to observe the polymerization of F-actin.Results The transfection of Cortactin-siRNA successfully inhibited the function of Cortactin.The relative content of MUC5AC was (0.210 ±0.013),(0.631 ±0.025),(0.473 ±0.112),(0.330 ±0.067),(0.272 ±0.019) in groups A,B,C,D and E,the group B was significantly higher than any other group (P =0.000,0.043,0.000,0.000).The Cortactin relative level in group B (0.670 ± 0.048)was significantly higher than that in group E (0.132 ±0.014) (P ＜0.01).But as compared with groups A,C,D (0.641 ±0.016,0.622 ±0.012,0.653 ±0.027),there was no significance (all P ＞0.05).The p-Cortactin relative level in group B (0.582 ± 0.067) was significantly higher than that in groups A,C,E (0.131±0.011,0.393±0.045,0.170±0.016) (P=0.000,0.021,0.000).But as compared with group D (0.511 ±0.029),there was no significance (P =0.246).Conclusion Rac-1,Cortactin and Factin are the main mediated molecules of airway shear stress-stimulated MUC5AC extracellular secretion.