【摘要】 目的:用β-escin在豚鼠心室肌细胞建立穿孔膜片箝技术（PPR）,并记录L-型钙电流(I_Ca,L),与传统的全细胞记录（WCR）模式相比较。方法:用酶消化法分离单个心室肌细胞,将β-escin配在电极内液中穿孔心室肌细胞膜形成PPR模式,用WCR及PPR技术记录心室肌细胞I_Ca,L,结果:β-escin 20,25,30μmol/L可在心室肌细胞膜穿孔形成PPR模式,用β-escin 25μmol/L成功率最高（16/17,94％）,用PPR模式记录的I_Ca,L其衰减明显比WCR方式记录的慢,I_Ca,L幅值在WCR形成后20min减小36％,而在PPR形成后30min仅缓慢减小8％,在两种模式下,I_Ca,L的I-V曲线,激活和失活曲线无显著差异,在PPR模式下,I_Ca,L的失活速率比在WCR模式下慢,在-20mV-+10mV电压下,其快失活相时间常数（τ_f）比WCR长（n=6,P<0.05）,在-10mV-+10mV电压下,其慢失活相时间常数（τ_s）也比WCR长（n=6,P<0.05）,在两种方式下,I_Ca,L的激活速率无显著差异。结论:用β-escin25μmol/L在豚鼠心室肌细胞能得到较稳定的PPR模式,此方法可以用于在普通的WCR模式下衰减较明显的电流如L-型钙电流的记录。更多还原

【Abstract】 AIM: To establish a perforated patch recording（PPR） mode with β-escin and compare L-type calcium current(I_Ca,L) recorded under PPR and normal whole-cell recording（WCR） condition in isolated guinea-pig ventricular myocytes. METHODS: Single myocytes were dissociated by enzymatic dissociation method. β-escin was added to the pipette solution to perforate the cell membrane and obtain PPR mode. I^Ca,L Was recorded using PPR and WCR techniques. RESULTS: β-Escin 20, 25, and 30 μmol/L could permeabilize the cell membrane and obtain PPR mode. With β-escin 25μmol/L, the success rate was highest（16/17, 94%） and the time required for permibilization was 2-15（8+4） min. Run-down of I^Ca,L was considerably slower in PPR than in WCR condition. The amplitude of I^Ca,L was decreased by 36% at 20 min after the formation of WCR, while it was slowly decreased by 8% at 30 min after the formation of PPR. The current-voltage relation（I-V） curves, activation and inactivation curves of I^Ca.L were not significantly different between WCR and PPR. The inactivation rate of I^Ca,L was slower in PPR than in WCR, the faster inactivation time constant（τ_f） was longer in PPR than in WCR at membrane potentials of-20 mV-+10 mV（n=6, P<0.05）, and the slower time constant（τ_s） was also longer in PPR than in WCR at membrane potentials of-10 mV-+10 mV （n=6, P<0.05）. There was no significant difference between the activation rate in WCR and PPR. CONCLUSION: Using β-escin 25 μmol/L can easily obtain stable PPR in isolated guinea-pig ventricular myocytes, and this method is useful in dealing with channels, which show run-down under normal WCR such as L-type Ca channel.更多还原