慢性缺氧对大鼠肺静脉平滑肌细胞内钙离子浓度的影响

Effect of chronic hypoxia on intracellular calcium ion concentration in rat pulmonary venous smooth muscle cells

目的:探索原代培养肺动脉高压模型大鼠肺静脉平滑肌细胞(PVSMCs)是否具有血管平滑肌功能及L型电压依赖性钙通道(VDCC)阻断剂硝苯地平对其细胞内钙离子浓度([Ca2+]i)的影响,为慢性缺氧性肺动脉高压发病机制的研究提供理论依据。方法:用显微镜操作和胶原酶消化法分离、培养肺动脉高压模型大鼠肺静脉平滑肌细胞,鉴定培养的细胞及计算纯度,利用细胞内钙离子浓度检测系统观察慢性缺氧对PVSMCs静息[Ca2+]i的影响及硝苯地平的干预作用。结果:该方法培养的肺动脉高压模型造模的PVSMCs呈典型的平滑肌特性,表现为"峰-谷"状生长,免疫化学α-Actin鉴定显色,而且培养纯度达到98%;慢性缺氧能使PVSMCs的静息[Ca2+]i从(104.3±7.9)nmol/L提高到(197.9±11.8)nmol/L(P<0.05);5 mmol/L的硝苯地平能完全阻断慢性缺氧PVSMCs的[Ca2+]i对高钾溶液的反应。结论:慢性缺氧可使PVSMCs的静息[Ca2+]i升高,其机制可能与激活PVSMCs的VDCC导致细胞外Ca2+内流有关。

Objective： To determine whether the primary culture of pulmonary venous smooth muscle cells（ PVSMCs） from rats with pulmonary hypertension exhibit functions of vascular smooth muscles,and effect of Ltype voltage-dependent Ca2 ＋channel（ VDCC） antagonist nifedipine on intracellular calcium ion concentration of PVSMCs,so as to provide scientific evidence for research on pathogenic role of chronic hypoxia in development of pulmonary hypertension. Methods： Microscopic operations and collagenase digestion were performed to isolate and culture the PVSMCs from rat model of chronic hypoxia. The PVSMCs were identified and studied for the purity of cultured cells. The effects of chronic hypoxia on resting [Ca2 ＋]iin PVSMCs and the interventional role of nifedipine were examined with In Cyte [Ca2 ＋]imeasurement system. Results： The PVSMCs from rat model of pulmonary hypertension showed typical features of smooth muscles as reflected by ＂peak and trough ＂growth pattern and positive immunohistochemical staining of α-actin. The purity of PVSMCs reached 98%. Resting[Ca2 ＋]iin PVSMCs was significantly elevated to（ 197. 9 ± 11. 8） nmol / L from 104. 3 ± 7. 9 nmol / L by chronic hypoxia（ P〈0. 05）. 5mmol / L Nifedipine was shown to completely block the PVSMC [Ca2 ＋]iresponse to KCL.Conclusion： Chronic hypoxia increases the resting [Ca2 ＋]iin rat PVSMCs probably via activation of VDCC in PVSMCs which in turn results in influx of extracellular Ca2 ＋.