上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

Wnt/β-catenin信号通路参与高渗条件下气道上皮细胞黏液高分泌

李 琪，陈贵华，周向东   

  1. 重庆医科大学附属第二医院呼吸内科， 重庆 400010
  • 出版日期:2014-05-28 发布日期:2014-05-30
  • 通讯作者: 周向东, 电子信箱: zxd999@263.net。
  • 作者简介:李 琪（1982—）， 女， 博士， 主治医师; 电子信箱: lqlq198210@sina.com。
  • 基金资助:

    国家自然科学基金（31171346，81100003，81370111）；重庆市自然科学基金（cstc2011jjA10046）；教育部博士点基金新教师类（20115503120006）；国家临床重点专科（2012-949）

Effects of Wnt/β-catenin pathway on the mucus hypersecretion of epithelial cells of airway under the hypertonicity condition

LI Qi, CHEN Gui-hua, ZHOU Xiang-dong   

  1. Department of Respiratory Medicine, the Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China
  • Online:2014-05-28 Published:2014-05-30
  • Supported by:

    National Natural Science Foundation of China，31171346, 81100003, 81370111； Chongqing Nature Science Foundation， cstc2011jjA10046； New Teacher Fund for Doctor Station，the Ministry of Education，China，20115503120006； National Key Clinical Special Foundation of China, 2012-949

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摘要：

目的 研究高渗刺激对人气道上皮细胞（16HBE）黏蛋白（MUC）5AC分泌的影响，以及Wnt/β-catenin信号通路可能的介导作用。方法 使用浓氯化钠配置高渗培养液，用高渗培养液刺激16HBE细胞，细胞分为常规培养（阴性对照组）以及高渗培养液刺激3、6、9、12 h组。ELISA法检测MUC5AC分泌量，Western blotting法检测人气道Wnt家族蛋白Wnt1、Wnt2、Wnt3a、Wnt4、Wnt5a、Wnt7a、Wnt10a、Wnt10b的表达情况。将Wnt4小片段siRNA转染16HBE细胞，再给予高渗培养液刺激。采用ELISA法检测培养上清的MUC5AC分泌量，采用Western blotting法检测细胞内经典Wnt/β-catenin通路相关信号分子的表达量，细胞免疫荧光法检测核因子（NF）-κB p65核转位情况。结果 高渗培养的16HBE细胞上清及胞质中检测到的MUC5AC分泌量显著高于阴性对照组，且MUC5AC分泌量在一定范围内呈时间依赖性（均P<0.05）。高渗刺激组Wnt1、Wnt2、Wnt3a、Wnt5a、Wnt7a、Wnt10a和Wnt10b表达量较阴性对照组无明显变化（均P>0.05），而Wnt4表达量显著升高（P<0.05）。高渗刺激组细胞内β-catenin和Cyclin D1的相对表达量显著高于阴性对照组，细胞免疫荧光法提示NF-κB p65核转位（均P<0.05）；而经转染Wnt4 siRNA后，高渗培养组上清及胞质内MUC5AC分泌量显著低于未转染的高渗培养组（P<0.05），细胞内β-catenin、CyclinD1水平及NF-κB p65的核转位现象也受到显著抑制（均P<0.05）。结论 高渗盐水可诱导16HBE细胞MUC5AC高分泌，Wnt/β-catenin信号通路在该效应中有重要的参与作用。

关键词: 高渗透压, 黏蛋白5AC, Wnt/β-catenin信号通路, 核因子κB

Abstract:

Objective To investigate the effects of hypertonicity on the secretion of Mucin (MUC) 5AC of 16HBE cells and possible mediate effects of Wnt/β-catenin pathway. Methods The hypertonic medium was prepared by the solution of high concentration of sodium chloride and the16HBE cells were stimulated by the hypertonic medium. The 16HBE cells were divided into the negative control group cultured in normal medium and groups stimulated by the hypertonic medium for 3, 6, 9, and 12 h. The secretion volume of MUC5AC was detected by the ELISA. The expressions of Wnt family proteins of human airway, i.e. Wnt1, Wnt2, Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt10a, and Wnt10b, were detected by the Western blotting. The 16HBE cells were transfected by the specific interference RNA (siRNA) of Wnt4 and stimulated by the hypertonic medium. The secretion volumes of MUC5AC in culture supernatants were detected by the ELISA. The expressions of signaling molecules related to classic Wnt/β-catenin pathway were detected by the Western blotting. The translocation of nuclear factor (NF)-κB p65 was visualized by the cell immunofluorescence. Results The secretion volumes of MUC5AC in supernatants and cytoplasm of 16HBE cells cultured by the hypertonic medium were significantly higher than those of the control group. The secretion volumes of MUC5AC exhibited a time-dependent manner in a certain range of concentration (P<0.05). Compared to the negative control group, the expression levels of Wnt1, Wnt2, Wnt3a, Wnt5a, Wnt7a, Wnt10a, and Wnt10b of the hyperosmotic groups were not significantly different (P>0.05), while the expression levels of Wnt4 significantly increased (P<0.05). The relative expression levels of β-catenin and Cyclin D1 in cells of the hyperosmotic groups were significantly higher than those of the negative control group. The cell immunofluorescence indicated that nuclear translocation of NF-κB p65 occurred (P<0.05). The secretion volumes of MUC5AC in supernatants and cytoplasm of the hyperosmotic groups that were transfected by the Wnt siRNA were significantly lower than those of the hyperosmotic groups that were not transfected by the Wnt siRNA (P<0.05). The levels of β-catenin and CyclinD1 and the translocation of NF-κB p65 in cells were also significantly inhibited (P<0.05). Conclusion The Wnt/β-catenin signaling pathway plays an essential role in the hypersecretion of MUC5AC in 16HBE cells induced by the hypertonicity.

Key words: osmotic pressure, mucin5AC, Wnt/β-catenin signaling pathway, nuclear factor-κB