慢性血栓栓塞性肺动脉高压患者肺血管平滑肌细胞膜电位的改变

Changes of Membrane Potential in Pulmonary Smooth Muscle Cell Isolated from Chronic Thromboembolic Pulmonary Hypertension Patients

目的探讨分离培养的慢性血栓栓塞性肺动脉高压患者肺血管平滑肌细胞的方法,观察其电生理学特性。方法分离慢性血栓栓塞性肺动脉高压(chronic thromboembolic pulmonary hypertension,CTEPH)患者内膜剥脱术后的肺组织标本(CTEPH患者组)和肺癌或肺大泡患者正常肺组织标本(正常对照组)的肺血管平滑肌细胞,并进行体外培养,用特异性抗体(smoothmuscle-α-actin,SM-α-actin)进行免疫荧光鉴定。膜片钳记录两组肺血管平滑肌细胞的静息膜电位和动作电位,分析比较两者的异同。结果①酶解法成功分离肺血管平滑肌细胞,经鉴定SM-α-actin阳性细胞达90%以上;②CTEPH患者组肺血管平滑肌细胞静息膜电位(-21.05 mV±2.20 mV,n=11)明显低于正常对照组(-38.12 mV±2.28 mV,n=10),细胞膜电位降低了约45%,处于明显去极化状态(P<0.001);③CTEPH患者组肺血管平滑肌细胞动作电位时程(action potential duration,APD):APD50(0.185 s±0.035 s),APD75(0.277 s±0.053 s),APD90(0.333 s±0.064 s)与正常对照组APD50(0.100 s±0.016 s),APD75(0.150 s±0.024 s),APD90(0.180 s±0.028 s)相比,均明显延长(P<0.05)。结论 CTEPH患者肺血管平滑肌细胞静息膜电位明显减小,动作电位时程延长,提示CTEPH患者肺血管平滑肌细胞存在明显的电生理学特性改变,该变化可能是细胞膜上电压依赖性钙离子通道激活、钙离子浓度增加、肺血管重构发生的重要因素。

Objective To investigate the method of isolating and culturing the pulmonary artery smooth muscle cells（PASMCs） from the endarterectomized specimens of CTEPH patients and to observe their electrophysiological characteristics. Methods The endarterectomized tissues of CTEPH patients undergoing pulmonary thromboendarterectomy（CTEPH-PAMSCs） and normal lung tissues of bronchogenic carcinoma or bullae patients（normal-PASMCs） were collected,the PASMCs were isolated by enzymatic method and identified by SM-α-actin.Patch clamp technique was used to measure the cell＇s membrane potential and action potential. ResultsHuman PASMCs from normal lung tissues and CTEPH patients＇ tissues were successfully isolated and cultured that was confirmed by SM-α-actin staining.Compared with PASMCs of normal control,the membrane potential in CTEPH-PASMCs was more depolarized（-38.12mV±2.28 mV,n=10 vs-21.05 mV±2.20 mV,n=11,P0.001） and the action potential duration at 50%,75%,90% were all prolonged（APD50： 0.100 s±0.016 s vs 0.185 s±0.035 s,APD75： 0.150 s±0.024 s vs 0.277 s±0.053 s,APD90： 0.180 s±0.028 s vs 0.333 s±0.064 s,P0.05）. Conclusion The results suggested that the PASMCs in CTEPH patients has a more depolarized resting membrane potential and a prolonged action potential duration,which might be important factors determining the calcium influx via the voltage-gated calcium channel in pulmonary vascular remodeling.