瞬时感受器电位通道在人脑微血管内皮细胞机械牵张中的作用及其信号转导机制

Effects of TRP channels on mechanical stretch of brain microvascular endothelial cells and its mechanism of signaling pathway

目的观察瞬时感受器电位(transient receptor potential,TRP)通道在人脑微血管内皮细胞(human brain microvascular endothelial cells,HBMEC)机械牵张刺激中的作用,并对其信号转导入胞内的机制进行初步探讨。方法 HBMEC体外培养,采用多功能小型生物撞击机给予机械牵张刺激,根据不同施加条件将细胞分为对照、单纯牵张、牵张+SKF-96365(非选择性TRP通道阻滞剂)、牵张+低分子量肝素(low molecular weight heparin,LMWH)、牵张+一氧化氮合酶抑制剂(N-nitro-l-arginine methyl ester,L-NAME)共5组。采用激光扫描共聚焦显微镜观察胞内游离Ca2+分布情况,流式细胞仪检测牵张前后胞内Ca2+荧光强度变化;Western blot检测牵张前后一氧化氮合酶(endothelial nitric oxide synthase,eNOS)蛋白表达情况。结果流式细胞仪检测结果显示,单纯牵张组和牵张+LMWH组细胞内游离Ca2+浓度较对照组显著升高(P<0.05);牵张+SKF-96365组胞内Ca2+浓度较单纯牵张组明显降低(P<0.05)。Western blot检测结果显示,单纯牵张组细胞eNOS蛋白表达水平明显增高(P<0.05);牵张+SKF-96365组和牵张+L-NAME组eNOS蛋白表达水平较单纯牵张组明显降低(P<0.05)。结论 HBMEC胞膜TRP通道可感知机械牵张刺激的激活,继而引起胞内游离钙离子浓度升高,其转导机制可能主要与胞外Ca2+内流相关。

Objective To observe the effects of transient receptor potential（ TRP） channels on stretch-induced injury of human brain microvascular endothelial cells（ HBMECs） and to explore its mechanism of signaling pathway. Methods After mechanical stretch by a mini-type multi-functional bio-impact machine,HBMECs were divided into a control group,a stretch group,a stretch ＋ SKF-96365 group,a stretch ＋ lowmolecular-weight heparin（ LMWH） group and a stretch ＋ N-nitro-l-arginine methyl ester（ L-NAME） group.The concentrations of free calcium ion in the cells before and after mechanical stretch were observed by confocal laser scanning microscopy and determined by flow cytometry. The expression of endothelial nitric oxide synthase（ eNOS） protein was detected by Western blotting. Results The concentration of free calcium ion detected by flow cytometry increased in the stretch group and stretch ＋ LMWH group（ P 0. 05）,and decreased in the stretch ＋ SKF-96365 group（ P 0. 05）. The expression of eNOS protein detected by Western blotting increased in the stretch group（ P 0. 05） and decreased in the stretch ＋ SKF-96365 group and stretch ＋ L-NAME group（ P 0. 05）. Conclusion The activation of TRP channels in HBMECs induced by mechanical stretch increases the free calcium concentration and the expression of eNOS protein. The mechanism may involve the influx of extracellular calcium ions.