细胞内钙浓度变化对心房肌小电导钙激活钾通道电流的影响

发布时间：2018-01-07 06:43

  本文关键词：细胞内钙浓度变化对心房肌小电导钙激活钾通道电流的影响　出处：《泸州医学院》2012年硕士论文　论文类型：学位论文

  更多相关文章： 小电导钙激活钾电流 胞内钙 L-型钙通道 心房颤动 人心房肌细胞 穿孔膜片钳技术

【摘要】：目的：心房纤颤(Atrial Fibrillation，AF)是最常见的快速性心律失常，随着年龄的增加其发病率增加,小于60岁的人群发病率为0.5％，大于80岁的人群发病率增加到10％。房颤可使死亡率增加1.5～1.8倍。AF患者可以产生明显的症状，例如丧失活动能力或使活动能力下降，且发生脑卒中的发病率增加到15％，心力衰竭的危险性明显增加。然而，目前房颤发生的机制仍未完全阐明。AF的发生与动作电位（Action potential，AP）的有效不应期（effective refractory period，ERP）缩短密切相关，而动作电位时程(action potential duration,APD)则由心肌细胞不同离子通道活动决定。因此，离子通道功能的变化可能与AF的发生及维持有关。小电导钙激活钾通道（small conductance Ca~(2+)-activated K~+channels, SK通道）是钙激活钾通道（Ca~(2+)-activated K~+channels, KCa）中的一种，具有电压不敏感、钙离子敏感的特性。近几年研究发现SK通道存在于心肌细胞上，且分子生物学证据表明在SK通道的4个亚型(SK1-4)中，SK_2通道在人和鼠的心房和心室表达存在差异，主要表达在心房。SK2通道为KCa通道，对胞内游离钙离子（[Ca~(2+)]i）高度敏感，发生反应迅速，可快速将细胞内钙离子浓度的变化转换成细胞膜电位变化。因此我们推测AF时胞内钙超载可能影响SK2通道功能，深入讨论这些问题对揭示AF发生的病理生理机制具有重要意义。本实验应用穿孔膜片钳技术（perforated Patch clamp，PPR）记录全细胞电流，观察窦性心律患者（Sinus Rhythm，SR）与AF心律患者心房肌细胞SK2通道电流变化及其对Ca~(2+)的敏感性的差异，以及观察L-钙通道的激动剂Bay-K8644、抑制剂维拉帕米（verapamil）对SK2通道电流的影响，探讨AF发生发展过程中可能出现的离子通道之间的相互作用，为AF机制的阐明提供更多的实验依据。方法：42例接受体外循环手术的患者分为两组：心房颤动组(AF)11例，窦性心律组（SR）31例。心肌细胞的获得：取体外循环术中切除的右心耳，在氧饱和Cardiplegic液中将组织剪成约1mm3的小块，EGTA脱钙，再进行两步酶消化：酶I(XXIV蛋白酶2-3U/ml，V胶原酶150U/ml，BSA1mg/ml)，酶II (V胶原酶150U/ml，BSA l mg/ml)。选取贴壁良好，折光性强，具有较强立体感，表面光滑的心肌细胞进行实验。SK2通道电流的记录：取细胞置于浴液中，用穿孔膜片钳技术记录全细胞电流，所得电流通过膜片钳放大器（CEZ-2300, Nihonkonden, Japan)放大，滤波（1KHz）后输入计算机，经Clampex10.0软件采集电流，Clampfit10.1、OriginPro8.0软件进行数据分析作图。实验分组：（1）以人心房肌细胞为研究对象，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，并用胞内钙成像系统验证穿孔前后胞内钙变化，以建立钙离子可以进入细胞内的穿孔膜片钳技术。（2）在全细胞穿孔膜片钳模式下观察SR组与AF组SK2通道电流的差异。（3）不同的胞内钙离子浓度对AF组与SR组SK2通道电流的影响。（4）全细胞穿孔膜片钳模式下观察L-型钙通道激动剂Bay-K8644、抑制剂（verapamil）对SK通道电流的影响。结果：（1）混合使用穿孔电极液6.88μg/ml β-escin和150μg/ml二性霉素B能形成稳定的穿孔膜片钳记录模式，且胞内钙测试系统检测可观察到穿孔后电极液至细胞内的游离钙离子浓度增加，F340/F380增强。（2）人心房肌细胞SK_2通道特性：①在全细胞穿孔膜片钳模式下，施以SK_2通道电流的刺激方案，可记录到一个内向整流混合电流，在浴液中加入SK_2通道的特异性阻断剂apamin，加药前后的内向整流综合电流相减即可获得apamin敏感的电流（SK2通道电流）。本实验记录到的人心房肌细胞SK2通道电流具有电压不敏感、内向整流的特性。②在全细胞穿孔膜片钳模式下，100nM apamin可以阻断部分内向整流混合电流，使内向整流混合电流减小，用浴液灌流将apamin洗脱后，被阻断的电流可以恢复。⑵SR组与AF组SK2通道电流的差异：在全细胞穿孔膜片钳模式下，电极液中钙离子浓度为5×10－7mol/L时，记录到的AF组的SK2通道电流明显大于SR组，尤其是在超极化方向。膜电位在-130mV时，SR组与AF组的SK2通道电流密度分别为：-2.92±0.35pA/pF、-6.83±0.19pA/pF（nSR=6，nAF=3，p＜0.05）。⑶不同浓度的胞内游离钙离子对SK2通道电流的影响：在电极液游离钙离子浓度为0mol/L、5×10~(－7)mol/L、10~(－6)mol/L膜电位为-130mV时， SR组SK2通道电流密度分别为-1.43±0.33pA/pF、-2.92±0.35pA/pF、-10.11±2.15pA/pF (n0=7，n_(5×10－7)=6，，n10－6=8，p＜0.05）；AF组SK_2通道电流密度分别为-2.17±0.40pA/pF、-6.83±0.19pA/pF、-14.47±2.89pA/pF (n0=4，n_(5×10－7)=3，n_(10－6)=4,p＜0.05）。（4）L-型钙通道与SK2通道电流的相关性研究：①L-型钙通道激动剂Bay-K8644对SK2通道电流的影响：在全细胞穿孔膜片钳模式下，在电极液游离钙离子浓度为5×10－7mol/L，膜电位为-130mV时，Bay-K8644能增大内向整流混合电流。②L-型钙通道抑制剂verapamil对SK2通道电流的影响：在全细胞穿孔膜片钳模式下，在电极液游离钙离子浓度为5×10~(－7)mol/L，膜电位为-130mV时，在浴液中加入verapamil的基础上，apamin对内向整流混合电流无抑制作用。结论：⑴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可形成稳定的全细胞记录模式，本技术方法即可保证胞内环境相对稳定，又可使电极液内的钙离子进入细胞内，从而改变细胞内的钙离子浓度，进而研究不同[Ca2+]i浓度对SK_2通道电流的调控。⑵电极液内游离钙离子在各浓度下(0mol/L、5×10~(－7)mol/L、10~(－6)mol/L)，AF组的SK2通道电流密度均大于SR组。⑶在AF状态下，SK2通道电流对Ca~(2+)的敏感性增强，与SR组比较，差异显著，提示SK_2通道电流可能参与了AF的发生发展。⑷L-型钙通道的激动剂Bay-K8644使SR组内向整流混合电流增大的趋势，在浴液中加入L-型钙通道的抑制剂verapamil时，apamin对内向整流混合电流无抑制作用，提示L-型钙通道与SK2通道之间存在着相关性。
[Abstract]:Objective: atrial fibrillation (Atrial Fibrillation AF) is the most common arrhythmia, with the increase of age, the incidence rate of incidence increased, less than 60 years old to 0.5% years old, more than 80 people to the increased incidence of atrial fibrillation in 10%. mortality could increase 1.5 ~ 1.8 times of.AF were significant the symptoms, such as loss of activity or the activity decreased, and the stroke incidence rate increased to 15%, the risk of heart failure increased significantly. However, the mechanism has not yet been completely elucidated AF occurrence and action potential of.AF (Action potential AP) the effective refractory period (effective refractory period. ERP) are closely related, and the action potential duration (action potential, duration, APD) is determined by the activity of myocardial cells of different ion channels. Therefore, changes in the function of ion channels may be related to the occurrence and maintenance of a AF . the small conductance calcium activated potassium channel (small conductance Ca~ -activated (2+) K~+channels, SK channel) is a calcium activated potassium channel (Ca~ (2+) -activated K~+channels, KCa) in one, with a voltage insensitive, calcium sensitive characteristics. In recent years, studies have found that SK channels are present in the myocardial cells, and the molecular biology evidence in 4 subtypes of SK channels (SK1-4), SK_2 channels are differentially expressed in human and rat atria and ventricles, mainly expressed in atrial.SK2 channels as KCa channels on intracellular free calcium ([Ca~ (2+)]i) highly sensitive reaction can rapidly. The rapid changes of the intracellular calcium concentration changes into cell membrane potential. So we speculate that AF intracellular calcium overload may affect the function of SK2 channels, in-depth discussion of these issues is of great significance to reveal the pathophysiological mechanisms of AF. The experimental application of perforated patch Clamp technique (perforated Patch clamp, PPR) to record the whole cell current, observation of patients with sinus rhythm (Sinus Rhythm, SR) and the changes of the SK2 channel AF in patients with atrial cardiac muscle cells and the current of Ca~ (2+) the difference of sensitivity, and the observation of L- calcium channel agonist Bay-K8644 inhibitor (verapamil) influence on Vera Pammy the SK2 channel current, the interaction between ion channels may appear on the occurrence and development of AF process, and provide more experimental basis for clarifying the mechanism of AF. Methods: 42 patients undergoing cardiopulmonary bypass surgery were divided into two groups: atrial fibrillation group (AF) in 11 cases, sinus rhythm group (SR) 31 cases. Cardiomyocytes were obtained: take the right atrial appendage resection during cardiopulmonary bypass, and cut into 1mm3 in Cardiplegic liquid oxygen saturation will be small, EGTA decalcification, and then a two step enzyme digestion enzyme I (XXIV protease 2-3U/ml, V collagenase 150U/ml, BSA1mg/m L II (V) enzyme, collagenase 150U/ml, BSA L mg/ml). Selected adherent, high refractive index, with a strong sense of three-dimensional, smooth surface of the myocardial cell experiments of.SK2 channel current records: cells were placed in a bath with the perforated patch clamp technique to record whole cell current, the current through the patch clamp amplifier (CEZ-2300 Nihonkonden, Japan), amplification, filtering (1KHz) after input to the computer by Clampex10.0 software to collect current, Clampfit10.1, OriginPro8.0 software to analyze the data mapping. Experimental groups: (1) in human atrial muscle cells as the research object, using amphotercin B and / or -escin beta as perforating electrode solution, perforated patch clamp experiment and intracellular calcium imaging system to verify the changes of intracellular calcium before and after perforation, in order to establish the calcium ions can enter the perforated patch clamp technique in cells. (2) in the whole cell perforated patch clamp mode was observed in the SR group and AF group SK2 The differences in channel current. (3) effects of different intracellular calcium ion concentration of AF group and SR group of SK2 channel current. (4) observed by patch clamp mode of L- type calcium channel agonist Bay-K8644 perforated whole cell inhibitor (verapamil) effect on SK channel current. Results: (1) mixed with perforation the electrode solution 6.88 g/ml beta -escin and 150 g/ml of amphotericin B can form a perforated patch recording mode is stable, and the intracellular calcium detection test system can be observed, the concentration of free calcium ion electrode solution to intracellular perforation after enhanced F340/F380. (2) human atrial muscle cell SK_2 channel characteristics: 1. In the whole cell perforated patch clamp mode, with the stimulus of SK_2 currents, recorded an inwardly rectifying mixed current SK_2 channel in the bath in a specific inhibitor of apamin, the inward rectifier current subtraction before and after comprehensive dosing can be obtained apamin sensitivity The current (SK2 current). Human atrial myocytes SK2 channel currents recorded in this experiment is insensitive to voltage characteristics of inward rectifier. The whole cell perforated patch clamp mode, 100nM apamin could block the inward rectifier part of mixed current, the inward rectifier hybrid current decreases, the apamin eluted by bath perfusion after current interruption can be restored. The difference between SR group and AF group: SK2 currents in whole cell perforated patch clamp mode, the electrode liquid calcium ion concentration of 5 * 10 7mol/L, SK2 channel current AF Group recorded significantly more than group SR, especially in the hyperpolarizing direction. The membrane potential at -130mV, SK2 channel current density of SR group and AF group respectively: -2.92 + 0.35pA/pF, -6.83 + 0.19pA/pF (nSR=6, nAF=3, P < 0.05). The different concentration of intracellular free calcium ion effect on SK2 channel current in the electrode liquid free calcium 绂诲瓙娴撳害涓

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