原代培养的大鼠远端肺动脉平滑肌细胞的功能研究

Function of primary culture of rat distal pulmonary arterial smooth muscle cells

目的探索原代培养的大鼠远端肺动脉平滑肌细胞(PASMC)是否具有血管平滑肌的功能,为缺氧性肺动脉高压等肺血管疾病发病机制及药物干预研究打下基础。方法采用显微操作和胶原酶消化法分离、培养大鼠远端PASMC,对培养细胞进行形态学观察、平滑肌α-actin免疫荧光染色鉴定,并用荧光显微镜法观测高钾(60 mmol·L-1KCl)溶液对大鼠远端PASMC的细胞内Ca2+浓度([Ca2+]i)的影响。结果培养的大鼠远端PASMC呈血管平滑肌细胞典型的"峰、谷"状生长,平滑肌α-actin免疫荧光染色呈阳性反应,60 mmol·L-1KCl溶液使PASMC的[Ca2+]i明显增高,5μmol·L-1硝苯地平能完全阻断PASMC的[Ca2+]i对KCl溶液的反应。结论原代培养的大鼠远端PASMC具有典型血管平滑肌细胞形态学、免疫学特征,具有功能正常的电压依赖性钙通道(VDCC),可广泛用于缺氧性肺动脉高压等肺血管疾病发病机制及药物干预的研究。

Aim To investigate the function of primary culture of rat distal pulmonary arterial smooth muscle cells（PASMC） for in vitro studies of the pathophysiology and medicine intervention of pulmonary circulation diseases,including hypoxic pulmonary hypertension.Methods Microsurgery operations and collagenase digestion were performed to isolate and culture rat distal PASMC.The rat distal PASMC were identified by morphological activity and immunofluorescence of smooth muscle α-actin.Furthermore,the response of intracellular Ca2＋ concentration（i） of PASMC to 60 mmol·L-1 KCl was tested by fluorescence microscope.Results The rat distal PASMC displayed a typical ＂peak and valley＂ growth pattern and smooth muscle α-actin showed a positive result.In addition,PASMC showed a clear increase in i in response to 60 mmol·L-1 KCl and nifedipine given at a 5 μmol·L-1 concentration completely blocked the i response to KCl in PASMC.Conclusion The rat distal PASMC show the morphological and immunological characteristics of vascular smooth muscle cells and possess the voltage-dependent Ca2＋ channels（VDCC） with normal functions.PASMC can be used to further explore the pathophysiological mechanisms and medicine intervention of pulmonary circulation diseases,including hypoxic pulmonary hypertension.