二性霉素B与β-escin混合穿孔电极液在人心房肌细胞SK2电流记录中的应用  

Application of recording SK2 current in human atrial myocytes by perforated patch clamp techniques with the mix of β-escin and amphotericin B

作　　者：王华[1] 李涛[1] 雷明[1] 李妙龄[1] 丁银元[1] 杨艳[1] 曾晓荣[1] 

机构地区：[1]泸州医学院医学电生理省部共建教育部重点实验室,四川泸州646000

出　　处：《中国应用生理学杂志》2012年第3期214-218,I0002,共6页Chinese Journal of Applied Physiology

基　　金：国家自然科学基金资助项目(30870903)

摘　　要：目的:为研究钙离子对人心房肌细胞小电导钙激活钾通道电流(ISK2)的调节作用,建立用二性霉素B(amphotericin B)与β-escin穿孔的膜片钳(PPR)技术。方法:体外循环术中取得右心耳,应用急性酶分离获得单个人体心房肌细胞,用二性霉素B和/或β-escin作为穿孔电极液,进行穿孔膜片钳实验,在此模式下测试钙离子对人心房肌细胞SK2电流的调控作用,并用胞内钙测试系统验证穿孔前后胞内钙变化。结果:混合使用穿孔电极液6.88μg/mlβ-escin和150μg/ml二性霉素B与单独用150μg/ml二性霉素B或6.88μg/mlβ-escin相比,前一种方法细胞容易封接,能形成稳定的穿孔膜片钳记录模式,可观察到SK2电流有激活,且胞内钙测试系统检测时可观察到穿孔后电极液至细胞内的游离钙离子浓度的增加,F340/380增强。结论:适当浓度的二性霉素B与β-escin混合使用进行穿孔膜片钳实验是一种稳定的全细胞膜片的记录技术,可以用于胞内游离钙离子对SK2电流调控的研究。Objective： To establish a perforated patch-clamp technology with amphotericin B and β-escin and to research the regulation of small conductance calcium-activated potassium channel SK2 currents by calcium ions. Methods： Single human atrial myocytes were enzymatically islolated from the right atrial appendage. Amphotericin B and / or β-esein were used by perforated electrode liquid. The regulation of SK2 current by calcium ions in human atrial myocytes was performed with the perforated patch-clamp technique. The intracellular calcium changes were measured by the intracellular calcium test system. Results： Mixed perforated electrode liquid compared with 150 μg/ml amphoteriein B or 6.88μg/ml β-escin alone, it was easy to seal eells and activate SK2 current by the former method. Moreover, the ration of F340/380 was consistent with the change of intracellular free calcium ion concentration increase after the formation of perforation. The ration of F340/380 was measured by intracellular calcium test system. Conclusion： The appropriate concentration of amphotericin B mixed with β-escin can form a stable whole-cell patch recording technology that is appropriate for the research of SK2 current regulation by intracellular calcium.

关 键 词：β-escin 二性霉素B 人心房肌细胞 小电导钙激活钾电流 穿孔膜片钳技术 

分 类 号：R331.3[医药卫生—人体生理学]