CAJ | 학술논문

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.
목적 탐토개민감수체(CaSR)재결양유도적기도점액고분비중적작용.방법 통과결양배양상(94％ N2、1％ O2、5％ CO2、37℃)배양인기도상피세포HBE16적방법복제세포결양모형.전염CaSR적소간우RNA (siRNA)급예선급여CaSR특이성격동제CaCl2、린지매C신호통로불동적억제제:Gαq/11단백특이성억제제(YM-254890),린지매C특이성억제제(U73122),삼린산기순수체(IP3R)특이성억제제(2-APB)급세포내Ca2+오합제(BAPTA-AM)후,각조세포재시이결양처리.채용반전록-PCR검측점단백(MUC)5AC mRNA적전록수평,Western인적법검측CaSR단백적표체수평,매련면역흡부(ELISA)법검측MUC5AC단백적상대함량,공취초현미경관찰세포내Ca2+농도적변화.결과 결양조CaSR단백상대표체수평(0.423 ±0.028)현저고우대조조(0.185 ±0.036)(t=12.75,P＜0.001);세포내Ca2+농도、MUC5AC단백상대함량급MUC5AC mRNA상대표체수평[(154.2±11.4) nmol/L、(0.624±0.063) μg/L、0.736±0.045]야현저고우대조조[(67.5±2.8)nmol/L、(0.257 ±0.051)tμg/L、0.321±0.034](t=18.04、11.06、18.05,균JP＜0.001).CaCl2능상조결양인기적상술작용,전염CaSR siRNA능구하조결양인기적상술효응(균P＜0.001).예선급여YM-254890、U73122、2-APB、BAPTA-AM처리세포,세포내Ca2+농도、MUC5AC단백상대함량급MUC5AC mRNA상대표체수평균현저저우단순결양조(균P＜0.05).결론 CaSR가통과Gαq/11/린지매C/삼린산기순/세포내Ca2+신호통로삼여결양유도적기도점액고분비.
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+].