中文摘要：

目的 探讨钙敏感受体（CaSR）在缺氧诱导的气道黏液高分泌中的作用.方法 通过缺氧培养箱（94％ N2、1％ O2、5％ CO2、37℃）培养人气道上皮细胞HBE16的方法复制细胞缺氧模型.转染CaSR的小干扰RNA （siRNA）及预先给予CaSR特异性激动剂CaCl2、磷脂酶C信号通路不同的抑制剂：Gαq/11蛋白特异性抑制剂（YM-254890）,磷脂酶C特异性抑制剂（U73122）,三磷酸肌醇受体（IP3R）特异性抑制剂（2-APB）及细胞内Ca2＋螯合剂（BAPTA-AM）后,各组细胞再施以缺氧处理.采用反转录-PCR检测黏蛋白（MUC）5AC mRNA的转录水平,Western印迹法检测CaSR蛋白的表达水平,酶联免疫吸附（ELISA）法检测MUC5AC蛋白的相对含量,共聚焦显微镜观察细胞内Ca2＋浓度的变化.结果 缺氧组CaSR蛋白相对表达水平（0.423 ±0.028）显著高于对照组（0.185 ±0.036）（t=12.75,P＜0.001）;细胞内Ca2＋浓度、MUC5AC蛋白相对含量及MUC5AC mRNA相对表达水平[（154.2±11.4） nmol/L、（0.624±0.063） μg/L、0.736±0.045]也显著高于对照组[（67.5±2.8）nmol/L、（0.257 ±0.051）tμg/L、0.321±0.034]（t=18.04、11.06、18.05,均JP＜0.001）.CaCl2能上调缺氧引起的上述作用,转染CaSR siRNA能够下调缺氧引起的上述效应（均P＜0.001）.预先给予YM-254890、U73122、2-APB、BAPTA-AM处理细胞,细胞内Ca2＋浓度、MUC5AC蛋白相对含量及MUC5AC mRNA相对表达水平均显著低于单纯缺氧组（均P＜0.05）.结论 CaSR可通过Gαq/11/磷脂酶C/三磷酸肌醇/细胞内Ca2＋信号通路参与缺氧诱导的气道黏液高分泌.

英文摘要：

Objective To explore the mechanism of calcium-sensing receptor （CaSR） in hypoxiainduced airway mucous hypersecretion.Methods Cultured human airway epithelial cells HBE16 by hypoxia incubator （94% N2,1% O2,5% CO2,37℃）.HBE16 were transfected with CaSR targeted small interfering RNA （CaSR-siRNA）,pretreated with a specific activator of CaSR CaCl2 and preincubated with various inhibitors [Gαq/11 protein inhibitor YM-254890,phospholipase C （PLC） inhibitor U73122,inositol 1,4,5-triphosphate receptors （IP3R） inhibitor 2-APB and cell-permeable intracellular calcium chelator BAPTAAM] before hypoxia.The level of MUC5AC mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）.And the relative content of CaSR protein was detected by Western blot.The relative content of MUC5AC protein was measured by enzyme-linked immunosorbent assay （ELISA）.The intracellular calcium concentration （[Ca2＋] i） was measured by laser confocal microscopy.Results The relative contents of [Ca2＋]i and MUC5AC protein were obviously higher in hypoxic group （（154.2 ± 11.4）nmol/L,（0.624 ±0.063） μg/L） than those in control group （（67.5 ± 2.8） nmol/L,（0.257 ± 0.051）μg/L） （all P 〈 0.01）.The relative levels of CaSR protein and MUC5AC mRNA were significantly higher in hypoxic group （0.423 ± 0.028,0.736 ± 0.045） than those in control group （0.185 ± 0.036,0.321 ±0.034） （all P 〈 0.01）.CaCl2 enhanced the effect of hypoxia.And the results had statistical significances （all P 〈0.01）.Transfection with CaSR-siRNA significantly decreased the effect of hypoxia （all P 〈0.01）.Pretreatments with Gαq/11 protein inhibitor,PLC inhibitor,IP3 R inhibitor or intracellular calcium chelator all significantly attenuated the hypoxia-induced MUC5AC hypersecretion and [Ca2＋]i （all P 〈 0.05）.Conclusion CaSR mediates hypoxia-induced airway mucous hypersecretion through a signaling pathway of Gαq/11/PLC/inositol 1,4,5-triphosphate （IP3） / [Ca2＋].