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肺动脉平滑肌细胞钾电流记录方法的建立

Establishment of recording potassium channel currents in pulmonary arterial smooth muscle cells

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【作者】 邝素娟钱卫民耿庆山邓春玉单志新林秋雄杨敏余细勇吴书林林曙光

【Author】 KUANG Su-juan, QIAN Wei-min, GENG Qing-shan, DENG Chun-yu, CHAN Zhi-xin, LIN Qiu-xiong, YANG Min, YU Xi-yong, WU Shu-lin, LIN Shu-guang (Medical Research Center of Guangdong General Hospital, Guangdong Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou 510080)

【机构】 广东省人民医院(医学研究中心)广东省医学科学院广东省心血管病研究所

【摘要】 目的在肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)上建立钙离子激活钾通道(calcium-activated potassium channel,KCa)电流、电压门控钾通道(voltage-gated potassium channel,KV)电流和内向整流钾通道(Inward rectifier channel,Kir)电流的记录方法。方法用急性酶解的方法分离出单个大鼠PASMC,利用全细胞膜片钳方法记录钾电流。结果①急性酶解分离得到高质量的单个大鼠PASMC,呈梭形,边界清楚,胞浆均匀透亮。②KV电流可以被5mmol/L4-AP明显抑制,使电流-电压关系曲线明显下移,在55mV时,KV电流密度从(133.86±7.36)pA/pF减少到(59.09±3.35)pA/pF(n=6,P<0.05)。③1mmol/L TEA对KCa电流有明显抑制作用,使电流-电压关系曲线明显下移,在55mV时,KCa电流密度从(9.03±1.42)pA/pF减少到(2.12±0.52)pA/pF(n=7,P<0.05)。④KATP电流可以被10μmol/L尼可地尔激活,使电流-电压关系曲线明显上移,在50mV时,Kv电流密度从(29.08±5.90)pA/pF增加到(88.90±7.98)pA/pF(n=10,P<0.05)。结论在肺动脉平滑肌细胞上成功地建立了KCa、KV和Kir电流的记录方法,可以为肺动脉相关疾病的发病机制和治疗方案的探索,提供有效的细胞模型基础。

【Abstract】 Objectives To establish the methods for recording calcium-activated potassium channel ( KCa) , voltage-gated potassium channel (KV) and inward rectifier channel (Kir) in pulmonary arterial smooth muscle cells (PASMC). Methods PASMC was acutely isolated by enzyme. Potassium currents were recorded by whole-cell patch- clamp technique. Results (1) High-quality single PASMCs were obtained. A typical single cell was fusiform shape and its cytoplasm was uniform and lucent. (2) KV currents were inhibited by 5 mmol / L 4-AP, Ⅰ-Ⅴ relation curve was shifted downward, KV current density were reduced from ( 133.86 ±7.36) pA / pF to ( 59.09 ±3.35) pA / pF (n=6, P<0.05) at 55 mV. (3) KCa currents were inhibited by 1 mmol/ L TEA. Ⅰ-Ⅴ relation curve was shifted downward. KV current density were reduced from (9.03±1.42) pA / pF to (2.12±0.52) pA / pF (n=7, P<0.05) at 55 mV. (4) KATP currents were activated by 10 μmol / L nicorandil. I-V relation curve was shifted upward. KV current density were increased from (29.08±5.90) pA / pF to (88.90±7.98) pA / pF (n=10, P<0.05) at 50 mV. Conclusions KCa, KV and Kir currents were recorded successfully in rat single PASMC, which offers an effective cell model to explore the pathogenesis of pulmonary artery diseases.

【关键词】 肺动脉肌细胞,平滑肌
【Key words】 pulmonary arterysmooth muscle cellpotassiuncalcium
【基金】 广东省医学科研基金资助项目(A2006002);广东省科技计划资助项目(2006B36002023);国家自然科学基金资助项目(30672077)
  • 【文献出处】 岭南心血管病杂志 ,South China Journal of Cardiovascular Diseases , 编辑部邮箱 ,2009年04期
  • 【分类号】R363
  • 【下载频次】88
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