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人气道上皮细胞感知冷刺激的受体机制

, PP. 2071-2076

Keywords: 气道上皮细胞,冷刺激,瞬时受体电位蛋白-,Ca+,PIP

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Abstract:

目的探讨温度感受器瞬时受体电位蛋白-8(transientreceptorpotentialmelastatin8,TRPM8)在人气道上皮细胞感受冷刺激过程中发挥的作用及其机制。方法用薄荷醇及冷空气(18℃)刺激人气道上皮16HBE细胞:①以TRPM8通道特异性拮抗剂BCTC、TRPM8shRNA为干预手段,动态观察在薄荷醇、冷刺激作用下细胞内Ca2+荧光强度变化来监测气道上皮细胞上TRPM8的功能;②以磷脂酰肌醇4,5-二磷酸(phosphatidylinositol4,5-bisphosphate,PIP2)荧光分子探针PLCδ1-PH-GFP转染细胞,通过动态观察各组细胞内PLCδ1-PH-GFP从细胞膜至细胞质的荧光转位变化来检测PIP2的动态变化来研究冷刺激作用下TRPM8的信号通路。结果①细胞内相对钙离子浓度变化:薄荷醇组处理4min后、冷刺激组处理6min后分别为(5.80±0.34)、(5.02±0.36),明显高于对照组(1.04±0.12)、(1.03±0.08)(P<0.05),1mmol/L薄荷醇组+BCTC组、1mmol/L薄荷醇组+转染TRPM8shRNA组最高值分别为(2.68±0.19)、(1.08±0.16),明显低于1mmol/L薄荷醇组,18℃+BCTC组、18℃+转染TRPM8shRNA组最高值分别为(1.77±0.22)、(1.02±0.18),明显低于18℃组(均P<0.05);②PLCδ1-PH-GFP从细胞膜至细胞质的转位变化(Fm/Fc值):1mmol/L薄荷醇组、冷刺激组最高值分别为(0.90±0.01)、(0.90±0.01),明显高于对照组最高值(1.01±0.02)(P<0.05),细胞处理200s时1mmol/L薄荷醇+BCTC15μmol/L组、1mmol/L薄荷醇组+转染TRPM8shRNA组分别为(0.86±0.03)、(?0.89±?0.02),明显低于1mmol/L薄荷醇组(0.36±0.06)(P<0.05),细胞处理220s时18℃+BCTC15μmol/L组、18℃+转染TRPM8shRNA组分别为(0.82±0.02)、(0.93±0.01),明显低于18℃组(0.40±0.06)(P<0.05)。结论TRPM8受体是人气道上皮细胞感受冷刺激的主要受体,并通过TRPM8→Ca2+→PLC→PIP2信号通路引起气道黏液高分泌等反应。

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