钙敏感受体通过ERK信号通路参与低氧诱导的人气道上皮细胞黏液高分泌  

Calcium-sensing receptor regulation of hypoxia-induced mucous hypersecretion by human airway epithelial cells through ERK signaling pathway

作　　者：杨红菊[1] 尤列.皮尔曼 维克多.科罗索夫 周向东[1] 

机构地区：[1]重庆医科大学附属第二医院呼吸内科,重庆400010 [2]俄罗斯医学科学院远东呼吸生理与病理研究所,俄罗斯布拉戈维申斯克675000

出　　处：《基础医学与临床》2014年第7期877-881,共5页Basic and Clinical Medicine

基　　金：国家自然科学基金(31171346);国家自然科学基金国际合作项目(31211120168)

摘　　要：目的探究钙敏感受体(Ca SR)在低氧诱导的气道黏液高分泌中的信号通路。方法低氧培养箱(37℃,94%N2-1%O2-5%CO2)培养人气道上皮细胞16HBE复制细胞低氧模型。分为对照组、低氧组、Ca SR特异性激动剂Ca Cl2+低氧组、对照siRNA+低氧组、Ca SR-siRNA+低氧组、细胞外信号调节激酶(ERK)信号通路特异性抑制剂U0126+低氧组。RT-PCR检测黏蛋白(MUC)5AC的转录水平,Western blot检测Ca SR、ERK1/2及磷酸化ERK1/2(p-ERK1/2)的蛋白相对含量,ELISA检测MUC5AC的分泌水平,四甲基偶氮唑盐法(MTT)测定细胞的活性。结果Ca SR表达于人气道上皮细胞,转染特异性的Ca SR-siRNA明显抑制Ca SR的表达。低氧组p-ERK1/2、MUC5AC mRNA和MUC5AC蛋白的相对含量分别为(0.63±0.11、0.51±0.03、0.56±0.07)较对照组(0.27±0.04、0.18±0.04、0.25±0.06)显著增加(P<0.05)。Ca SR的激动剂Ca Cl2可进一步增强低氧引起的上述作用(P<0.05),而转染Ca SR-siRNA及给予ERK信号通路特异性抑制剂U0126可抑制低氧引起的上述效应(P<0.05)。结论 Ca SR可通过MEK/ERK1/2信号通路参与低氧诱导的气道黏液高分泌。Objective To explore the signal pathway of calcium-sensing receptor （CaSR） in hypoxia-induced airway mucous hypersecretion.Methods Human airway epithelial cells 16HBE were incubated in hypoxia incubator （37 ℃,94% N2-1% O2-5% CO2） and divided into 6 groups：control group,hypoxia group,CaSR specific activator CaCl2 group treated with hypoxia,negative-siRNA control group treated with hypoxia,CaSR-siRNA group treated with hypoxia,extracellular signal regulated kinase（ERK） specific inhibitor U0126 group treated with hypoxia.The level of MUC5AC mRNA were detected by RT-PCR.The protein levels of CaSR,ERK and phosphorylated ERK （p-ERK） were detected by Western blot.The level of MUC5AC secretion was measured by ELISA.Cell survival rate was measured by MTT assay.Results CaSR protein was expressed in 16HBE and transfection with CaSR-siR-NA significantly inhibited the expression of CaSR.In hypoxia group,the level of p-ERK,MUC5AC mRNA and MUC5AC protein increased （0.63 ±0.11,0.51 ±0.03,0.56 ±0.07） versus the control group （0.27 ±0.04,0.18 ± 0.04,0.25 ± 0.06） （P 〈 0.05）.CaCl2 （a CaSR agonist） amplified the effect of hypoxia （P 〈 0.05）.Transfection with CaSR-siRNA or pretreatment with ERK inhibitor decreased the level of p-ERK,MUC5AC mRNA and MUC5AC protein induced by hypoxia （P 〈 0.05）.Conclusions CaSR regulates hypoxia-induced airway mucous hypersecretion through MEK/ERK1/2 signaling pathway.

关 键 词：钙敏感受体 缺氧 黏蛋白 ERK 

分 类 号：R363.1[医药卫生—病理学]