【作者】 周彤；

【导师】 曾秋棠；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 第一部分家兔耐钙心肌细胞急性分离及电生理记录的方法学探讨目的:细胞标本制备的质量是膜片钳实验成功的关键,本研究探讨通过改变细胞外液成分获取适于电生理研究的耐钙心室肌细胞及L-型钙通道电流(I_Ca-L)、延迟整流钾电流（I_K）记录的方法。方法:家兔心室肌细胞急性分离采用Langendorff灌流装置及酶解消化分离技术,心脏灌流前实验组细胞外液为正常台氏液,对照组细胞外液为无钙台氏液,观察细胞外液中的钙离子对获取适于电生理记录的单个耐钙心肌细胞的影响;应用全细胞膜片钳技术,实验组分别以含BaCl₂的台氏液及N-甲基-D-葡萄糖胺（NMDG）溶液作为细胞外液记录I_Ca-L及I_K,对照组为正常台氏液。结果:与对照组相比较,实验组心室肌细胞存活率和耐钙心肌细胞存活率均明显升高（71.0%±9.7% vs 54.5%±10.1%; 52.0%±7.9% vs 38.5%±10.8%, n=10, P<0.05）。实验组引出的I_Ca-L峰值电流大于对照组（15.2±7.1 pA/pF vs 7.4±2.4 pA/pF, n=10,P<0.05）。实验20 min与实验5 min记录的I_Ca-L峰值比较,对照组I_Ca-L峰值电流的变化率为36.1%±9.1% （n=10, P<0.05）,即I_Ca-L峰值电流在实验20 min衰减;实验组I_Ca-L峰值电流的变化率为3.0%±4.7% （n=10, P>0.05）,即I_Ca-L峰值电流幅值稳定。实验组I_K时间依赖性外向电流及尾电流随去极化时间延长增大作用更明显（1.39±0.05pA/pF vs 0.53±0.14 pA/pF, 0.74±0.09 pA/pF vs 0.39±0.13 pA/pF, n=10, P<0.05）。结论:细胞外液中的钙离子有助于获取耐钙心肌细胞,以含BaCl₂的台氏液及NMDG溶液作为细胞外液易于获得典型而稳定的I_Ca-L及I_K。第二部分阿魏酸钠与胺碘酮对家兔心室肌细胞L-型钙通道电流的影响目的:探讨阿魏酸钠对家兔心室肌细胞膜L-型钙通道电流(I_Ca-L)的影响。方法:酶解法急性分离家兔单个心室肌细胞,以经典的Ⅲ类抗心律失常药物胺碘酮为对照,应用膜片钳全细胞记录技术观察3、10、30、100μmol/L的阿魏酸钠及1、3、10、30μmol/L的胺碘酮对心室肌细胞膜I_Ca-L的作用。结果:阿魏酸钠及胺碘酮均呈浓度依赖性抑制L-型钙电流。3、10、30、100μmol/L的阿魏酸钠对I_Ca-L的抑制率分别为11.1%±2.4%、26.9%±6.2%、40.5%±5.0%、61.9%±5.5% （P<0.05）;1、3、10、30μmol/L的胺碘酮对I_Ca-L的抑制率分别为21.1%±3.8%、32.6%±2.6%、52.6%±4.6%、71.4%±7.0% （P<0.05）;半数抑制浓度(IC₅₀)分别为32.6μmol/L及9.5μmol/L,阿魏酸钠的抑制作用弱于胺碘酮（P<0.05）。阿魏酸钠对I_Ca-L动力学的影响类似于胺碘酮,二者均能使I_Ca-L电流-电压曲线上移,未改变I-V曲线形状;使稳态激活曲线右移,失活曲线左移,使激活曲线与失活曲线之间相交的“窗流”区域缩小;并使I_Ca-L恢复曲线的t值增加,恢复曲线右移,即减慢钙通道灭活后的恢复过程。结论:阿魏酸钠对I_Ca-L具有浓度依赖性阻滞作用,且使I_Ca-L的激活减慢、失活加快,并且失活后的恢复时间延长,阿魏酸钠的抑制作用虽弱于胺碘酮,但具有与胺碘酮相似的阻滞I_Ca-L且影响I_Ca-L的动力学特性,可能是其抗心律失常作用的电生理机制之一。第三部分阿魏酸钠与胺碘酮对家兔心室肌细胞钾通道电流的影响目的:探讨阿魏酸钠对家兔心室肌细胞膜延迟整流钾电流快速与缓慢激活成分(I_Kr、I_Ks)、内向整流钾电流(I_Kl)、瞬时外向钾电流(I_to)的影响。方法:酶解法急性分离家兔单个心室肌细胞,以经典的Ⅲ类抗心律失常药物胺碘酮为对照,采用膜片钳全细胞记录技术观察浓度为3、10、30、100μmol/L的阿魏酸钠对心室肌细胞膜I_Kr、I_Ks、I_Kl及I_to的作用。结果:阿魏酸钠的作用弱于胺碘酮,二者均可浓度依赖性抑制I_Kr、I_ks时间依赖性外向电流及尾电流(I_Kr,tail、I_{Ks, tail}),3、10、30、100μmol/L的阿魏酸钠对I_{Kr, tail}的抑制率为12.1%±2.5%、24.1%±3.0%、47.0%±5.8%及58.5%±8.3%（n=5,P<0.05）;对I_{Ks, tail}的抑制率为15.6%±6.4%、27.1%±6.5%、45.6%±5.7%及51.8%±6.6%（n=5,P<0.05）,其对I_{Kr, tail}及I_{Ks, tail}的半数抑制浓度(IC₅₀)均大于胺碘酮（43.6 vs 3.48μmol/L,44.8 vs 5.11μmol/L）。30、100μmol/L的阿魏酸钠及10、30μmol/L的胺碘酮可使I_kl的I-V曲线左移,在-100 mV及-20 mV测试电压下,浓度为100μmol/L的阿魏酸钠对I_kl内向电流及外向电流抑制率小于30μmol/L的胺碘酮（24.3%±8.8% vs 29.7%±8.0%; 17.7%±3.3% vs 20.1%±10.6%, n=5, P<0.05）。阿魏酸钠与胺碘酮均不影响I_to及其I-V曲线。结论:阿魏酸钠对复极期钾电流具有阻滞作用,可能是其抗心律失常作用的电生理机制之一。第四部分阿魏酸钠与胺碘酮对家兔心室肌细胞钠通道电流的影响目的:探讨阿魏酸钠对家兔心室肌细胞膜钠通道电流(I_Na)的影响。方法:酶解法急性分离家兔单个心室肌细胞,以经典的Ⅲ类抗心律失常药物胺碘酮为对照,采用膜片钳全细胞记录技术观察浓度为100μmol/L的阿魏酸钠及10μmol/L胺碘酮对心室肌细胞膜I_Na的作用。结果:10μmol/L胺碘酮使I_Na峰值电流由20.9±3.63 pA/pF减为12.0±2.69 pA/pF（n=5, P<0.05）,抑制率为42.6%±4.87%;胺碘酮使I_Na的I-V曲线上移。100μmol/L阿魏酸钠不改变I_Na峰值电流（16.6±4.52 pA/pF vs 15.8±3.21 pA/pF,抑制率为6.12%±5.41%, n=5, P>0.05）,且不影响I-V曲线。结论:阿魏酸钠急性灌流不影响家兔心室肌细胞I_Na。更多还原

【Abstract】 Part one Investigation of the methods for acute isolatingcalcium-tolerant ventricular myocytes andelectrophysiolological recordingsObjective:It is important to obtain viable cells for long period of electrophysiologicalrecording.In this study,we explore the influences of ingredients of extracellularsuperfusate on acute isolation of calcium-tolerant ventricular myocytes and recording ondelayed rectifier potassium （I_K） and L-type calcium current (I_Ca-L).Methods:Single ventricular myocytes of rabbits were obtained enzymatically byretrograde Langendorff perfusing apparatus.Before Langendorff perfusing,theextracellular superfusate was Tyrode’s solution in experiment group and calcium-freeTyrode’s solution in control group.The effect of extracellular superfusate Ca²⁺ oncalcium-tolerant ventricular myocytes available for electrophysiological recordings wereinvestigated.Whole-cell patch clamp recording technique was used to record the changes ofI_K and I_Ca-L in experimental groups following the administration of extracellular with BaCl₂and N-methyl-D-glu-camine （NMDG） in experiment group and extracellular Tyrode’ssolution in control group.Results:The survival rates of ventricular myocytes and calcium-tolerant myocyteswere significantly higher in experimental groups than in control group （71.0%±9.7% vs54.5%±10.1%,52.0%±.7.9% vs 38.5%±10.8%,n=10,P＜0.05）.In experimental group, peak I_Ca-L was elicited stably and larger than in control group （15.2±7.1 pA/pF vs 7.4±2.4pA/pF,n=10,P＜0.05）.As compared 20 min with 5 min,the rate of change in peak I_Ca-L was36.1%±9.1% in control group （n=10,P＜0.05）,and 3.0%±4.7% in experimental group（n=10,P＞0.05）.The time-dependent outward current and tail current of I_K were higher inexperimental group than in control group （1.39±0.05 pA/pF vs 0.53±0.14 pA/pF,0.74±0.09 pA/pF vs 0.39±0.13 pA/pF,n=10,P＜0.05）.Conclusion:Extracellular superfusate Ca²⁺ is helpful for isolating singlecalcium-tolerant cardiomyocytes.With BaCl₂ and NMDG in extracellular superfusate,theionic currents of I_Ca-L and IK were elicited typically and easily.Part two Effects of sodium ferulate and amiodarone on L-typecalcium channel current in isolated rabbit ventricular myocytesObjective:To study the effect of sodium ferulate on L-type calcium current (I_Ca-L) inisolated rabbit ventricular myocytes.Methods:Compared with amiodarone- a typical classⅢantiarrhythmic drug,singleventricular myocytes of rabbits were isolated enzymatically and whole-cell patch clamprecording technique was used to record the changes of I_Ca-L following the administration ofsodium ferulate at 3,10,30 and 100μmol/L.Results:Both sodium ferulate and amiodarone could block the I_Ca-L which hadconcentration dependence,however,sodium ferulate inferior to amiodarone （P＜0.05）.PeakI_Ca-L was reduced 21.1%±3.8%,32.6%±2.6%,52.6%±4.6% and 71.4%±7.0% （n=5,P＜0.05） respectively at amiodarone 1,3,10 and 30μmol/L.In contrast,Peak I_Ca-L wasreduced 11.1%±2.4%,26.9%±6.2%,40.5%±5.0% and 61.9%±5.5% （n=5,P＜0.05）respectively at sodium ferulate 3,10,30 and 100μmol/L.IC₅₀ （the concentration for half-maximal block） was 32.6μmol/L and 9.5μmol/L.The current-voltage curve movedupward,stead-state activated curve moved right and stead-state deactivated curve movedleft,furthermore,they delayed the recovery time course of I_Ca-L from inactive state. Conclusion:Sodium ferulate block L-type calcium channel,slow it’s activate,rapidit’s inactivate and delay the recovery time.These may be the related mechanisms of theantiarrhythmic effects.Part three Effects of sodium ferulate and amiodarone on potassiumchannel currents in isolated rabbit ventricular myocytesObjective:To study the effect of sodium ferulate on rapidly and slowly activatingcomponent of delayed rectifier potassium current (I_Kr,I_Ks),inwardly rectified potassiumcurrent (I_K1) and transient outward potassium current (I_to)in isolated rabbit ventricularmyocytes.Methods:Compared with amiodarone - a typical classⅢantiarrhythmic drug,singleventricular myocytes of rabbits were isolated enzymatically and whole-cell patch clamprecording technique was used to record the changes of I_Kr,I_Ks,I_K1 and I_to following theadministration of sodium ferulate at 3,10,30 and 100μmol/L.Results Sodium ferulate was inferior to amiodarone,though both of them could blockthe time-dependent outward current and tail current of I_Kr and I_Ks which had concentrationdependence.Peak tail currents of I_Kr were reduced 12.1%±2.5%,24.1%±3.0%,47.0%±5.8%,58.5%±8.3% （n=5,P＜0.05） respectively,while,peak tail currents of I_Ks werereduced 15.6%±6.4%,27.1%±6.5%,45.6%±5.7%,51.8%±6.6% （n=5,P＜0.05）respectively at sodium ferulate 3,10,30 and 100μmol/L.IC₅₀ （the concentration for half-maximal block） were higher than amiodarone respectively （43.6 vs 3.48μmol/L,44.8 vs5.11μmol/L）.Compared with amiodarone （30μmol/L）,sodium ferulate （100μmol/L）decreased I_k1 by 24.3%±8.8% vs 29.7%±8.0% at test potential of -100 mV （n=5,P＜0.05）,At -20mV,I_K1 decreased by 17.7%±3.3% vs 20.1%±10.6% （n=5,P＜0.05）.Thecurrent-voltage curve of I_K1 moved left by sodium ferulate （30,100μmol/L） andamiodarone （10,30μmol/L） acute superfusion.However,neither of them alter I_to and it’scurrent-voltage curve. Conclusion:Sodium ferulate block potassium channels,these may be the relatedmechanisms of the antiarrhythmic effects.Part four Effects of sodium ferulate and amiodarone on sodiumchannel currents in isolated rabbit ventricular myocytesObjective:To study the effect of sodium ferulate on sodium current (I_Na) in isolatedrabbit ventricular myocytes.Methods:Compared with amiodarone- a typical classⅢantiarrhythmic drug,singleventricular myocytes of rabbits were isolated enzymatically and whole-cell patch clamprecording technique was used to record the changes of I_Na.Results:The amplitude of peak I_Na was decreased by acute amiodarone （30μmol/L）superfusion （20.9±3.63 pA/pF vs 12.0±2.69 pA/pF,n=5,P＜0.05）,the inhibition was42.6%±4.87%,and current-voltage curves of I_Na moved upward.However,sodiumferulate （100μmol/L） superfusion had no effect on I_Na （16.6±4.52 pA/pF vs 15.8±3.21pA/pF,n=5,P＜0.05 ）,the inhibition was 6.12%±5.41%,neither did sodium ferulate altercurrent-voltage curves of I_Na （ n=5,P＞0.05 ）.Conclusion:Sodium ferulate had no effect on sodium channel current in Langendorff-perfused rabbit heart.更多还原