实验服务
详细说明1 Test Method1.1 Treatment GroupThree (3) concentrations of the Test Article be applied sequentially in ascending concentration to each Purkinje fiber. Each concentration will be tested in at least four (4) fibers (n ³ 4). The test article Vehicle Control will be applied to at least four (4) fibers (n ³ 4) with exposure times to approximate those of the test group. At the end of the vehicle exposure period the Positive Control article (sotalol at 100 µM) will be applied. 1.2 Heart preparation Rabbits will be purchased from Xiehe Jianhao (Beijing) in this study. Heparin will be given (500 U/kg i.v.) followed by sodium pentothal (20-40 mg/kg, i.v.). The skin of the entire chest area will be shaved. After anesthesia is confirmed, the heart will be removed rapidly through a left lateral thoracotomy. The heart will be immediately placed into cold, oxygenated cardioplegic Tyrodes solution (modified to contain 26 mM KCl). The heart will be placed in a large glass beaker and flushed with Tyrode’s solution to remove blood. 1.3 Electrophysiological ProceduresA Purkinje fiber will be placed in a recording chamber mounted on a heated platform, and superfused at approximately 2 mL /min with vehicle control solution. The bath temperature will be controlled using a water bath chamber. Bath temperature will be measured using a thermistor probe. Intracellular membrane potentials will be recorded using conventional intracellular microelectrodes pulled from borosilicate glass capillary tubing on a micropipette puller (Sutter Instruments P-97, Novato, CA) filled with 3 M KCl solution. Action potentials will be evoked by repetitive electrical stimuli (1-3 ms duration, approximately 1.5 times threshold amplitude). A bipolar, insulated (except at the tip) electrode will be used to deliver pulses generated by a photo-isolated, electronic stimulator. Analog signals will be low-pass filtered at 10 kHz before digitization at 50 kHz, and stored on hard disk using a PC-compatible computer controlled by Axon Pclamp 10.2 software.1.4 Test Procedures Concentration-response and rate-dependence will be determined by the following test procedure (summarized in the schedule table below). Purkinje fibers will be paced continuously at a basic cycle length (BCL) for at least 30 minutes (recovery and stabilization) before obtaining control AP responses. Only fibers with resting potentials more negative than –80 mV and normal AP morphology will be used. After recovery and stabilization, the control solution will be applied for at least 5 minutes for equilibration in the absence of test article and the fiber will be pulsed at a basic cycle length of 2 s. At the end of this period, baseline control APD rate-dependence will be measured using stimulus pulse trains consisting of approximately 50 pulses at BCL of 2, 1 and 0.5 s. For test group, after returning to BCL of 2 s, test article at the lowest concentration will be applied for at least 20 minutes to allow equilibration, and the stimulus trains repeated. The entire sequence (~20 minutes of equilibration followed by stimulus trains at decreasing BCL) will be repeated at cumulatively increased test article concentration. For vehicle control group, entire sequence will be repeated with time-matched vehicle controls. The sequence for the positive control will be repeated at end of vehicle controls. The average responses from the last five recorded action potentials from each stimulus train will be analyzed for each test condition. Stimulus and Solution Application Schedule SolutionApproximateDuration (min)MeasurementStimulus Interval, BCL (s)Vehicle Control Solution³ 30Recovery and Stabilization0.5 - 2Vehicle Control Solution³ 5APD rate dependence2Vehicle Control Solution~ 3APD rate dependence 2, 1, 0.5Test article concentration or Vehicle Control (1·· X)³ 20Equilibration2Test article concentration or Vehicle Control (1·· X)~ 3APD rate dependence 2, 1, 0.5100 µM Sotalola³ 20Equilibration 2100 µM Sotalola~ 3APD rate dependence 2, 1, 0.5a Following the final vehicle group only(1·· X): Repeated for each test article concentration; X = number of test article concentration to be applied and tested on each fiber.1.5 Electrophysiological Data Analysis1.5.1 Action Potential AnalysisData will be analyzed with Pclamp 10.2 software. The following parameters will be determined: RMP (resting membrane potential, mV), APA (action potential amplitude, mV), Vmax (maximum rate of rise V/s), APD60 and APD90 (action potential duration at 60 and 90% repolarization, respectively, ms). Concentration-response data will be presented relative to baseline before test article application. APD60, APD90 and Vmax at each stimulus frequency will be presented as percent change (D%) from baseline at each concentration. RMP and APA data will be presented as change in membrane potential (DmV).1.5.2 Statistical AnalysisData will be reported as Mean ± SEM. Pooled data will be tabulated for each condition: control baseline, test article concentration and stimulus frequency. Changes in action potential parameters will be evaluated using one-way ANOVA followed by Dunnett’s multiple comparison test to determine whether the change from baseline observed after equilibration in each test article concentration is significantly different (P<0.05) from that observed in the time-matched vehicle control group.
CFDA药物安全药理学研究技术指导原则中，指出采用离体动物或人心肌细胞、培养心肌细胞系或克隆的人离子通道的异种表达体系测定离子流。豚鼠乳头肌动作电位检测为药物安全性评价提供更多的参考指标。
实验提供全部的原始数据及分析的结果
详细说明表达各种钠离子通道亚型的稳定细胞系信息如下 ：钠通道稳定表达的HEK293细胞系在含有10% 胎牛血清及1.2mg/ml G418的DMEM培养基中培养，培养温度为37℃，二氧化碳浓度为5%。细胞传代：除去旧培养基并用PBS洗一次，然后加入1 ml TrypLE? Express溶液，37°C孵育1分钟。当细胞从皿底脱离，加入5 ml 37°C预热的完全培养基。将细胞悬液用吸管轻轻吹打使聚集的细胞分离。将细胞悬液转移至无菌的离心管中，1000rmp离心5分钟收集细胞。扩增或维持培养，将细胞接种于10厘米细胞培养皿，每个细胞培养皿，接种细胞量为3.5*105 cells（最终体积：10 ml）。为维持细胞的电生理活性，细胞密度必须不能超过80%。膜片钳检测，实验之前细胞用TrypLE™ Express分离，将3*103 细胞铺到盖玻片上，在24孔板中培养（最终体积：500μl），18个小时后，进实验检测。电生理检测方法 用微电极拉制仪（P97，Sutter Instruments）将毛细玻璃管（BF150-86-10，Sutter Instruments）拉制成记录电极。在倒置显微镜（IX71，Olympus）下操纵微电极操纵仪（MP285，Sutter Instruments）将记录电极接触到细胞上，给予负压抽吸，形成GΩ封接。形成GΩ封接后进行快速电容补偿，然后继续给予负压，吸破细胞膜，形成全细胞记录模式。然后进行慢速电容的补偿并记录膜电容及串联电阻。不给予漏电补偿。当全细胞记录的电流稳定后开始给药，每个药物浓度作用至5分钟后检测下一个浓度，在记录期间独立重复检测多个细胞。所有电生理实验均在室温下进行。实验数据由 EPC-10 放大器(HEKA)进行采集并储存于PatchMaster (HEKA)软件中。检测实验的刺激方案：数据质量标准 (1) 串联电阻 ≤ 20 MΩ；(2) 封接电阻 ≥ 1 GΩ；(3) 起始电流幅度适宜；(4) 电流没有明显的自发性衰减(5分钟内自发性衰减小于5%)；(5) 在膜电位为-80mV下无明显的漏电流 (漏电流 ≤ 100 pA)。数据分析首先将每一个药物浓度作用后的电流和空白对照电流标准化(),然后计算每一个药物浓度对应的抑制率(1-)。对每一个浓度计算平均数和标准误， 并用以下的方程计算每种化合物的半抑制浓度： 用以上方程对剂量依赖效应进行非线性拟合， 其中c代表药物浓度，IC50为的半抑制浓度，h代表希尔系数。曲线拟合以及IC50的计算利用IGOR软件完成。NaV1.1ChannelNav1.1GeneSCN1ASourceshumanCatalog Ref.ICE-CHO-Nav1.1 + β1Expression systemCHOMethodwhole cell patch clampReference inhibitorTTX (5.6nM±0.8nM)DistributionCNS and cardiac myocytesRelated diseasesPain, Epilepsy, Anxiety, Depression Related, Degenerative Diseases, Dravet syndrome, West syndrome, familial autism NaV1.2ChannelNaV1.2GeneSCN2ASourceshumanCatalog Ref.ICE-HEK-Nav1.2Expression systemHEK293Methodwhole cell patch clampReference inhibitorTTX (11.7±0.783nM)TargetPain, Seizure, Epilepsy, Anxiety, Depression Related, Degenerative Diseases NaV1.3ChannelNav1.3GeneSCN3ACatalog Ref.ICE-HEK-Nav1.3SourceshumanExpression systemHEK293Methodwhole cell patch clampReference inhibitorTTX (5.0±0.54nM)TargetPain, Epilepsy, Anxiety, Depression Related NaV1.5ChannelNaV1.5GeneSCN5ACatalog Ref.ICE-HEK-Nav1.5SourceshumanExpression systemHEK293Methodwhole cell patch clampReference inhibitorTTX (1.0μM± 438.7nM)TargetBrugada syndrome, long QT syndrome, progressive cardiac conduction disease ,dilated cardiomyopathy, sick sinus syndrome, and atrial fibrillation NaV1.6ChannelNav1.6GeneSCN8ACatalog Ref.ICE-HEK-Nav1.6SourceshumanExpression systemHEK293Methodwhole cell patch clampReference inhibitorTTX (1.9±0.302nM)TargetEpilepsy, Degenerative Diseases, NaV1.7ChannelNav1.7GeneSCN9ACatalog Ref.ICE-HEK-Nav1.7SourceshumanExpression systemHEK293Methodwhole cell patch clampReference inhibitorTTX (1.3±0.508nM)TargetPain, Anxiety, Depression Related, erythromelalgia NaV1.8 ChannelNaV1.8GeneSCN10ACatalog Ref.ICE-HEK-Nav1.8SourceshumanExpression systemHEK293Methodwhole cell patch clampReference inhibitorTTX (2.90±0.803nM)TargetPain, Anxiety, Depression Related
1. L-type calcium channel (CaV1.2)ChannelCav1.2/β2/α2/δ1, L-typeCatalog Ref.ICE-CHO-Cav1.2GeneCACNA1CSourceshumanExpression systemCHOMethodwhole cell patch clampReference InhibitorNifedipine(31.3nM±6.8nM), verapamilTargetTimothy syndrome, long QT syndrome, Pain, epilepsy, hypertension, stroke, arrhythmia, Autism 2、N-type calcium channel (CaV2.2)ChannelCav2.2/β3/α2δ1 , N-typeCatalog Ref.ICE-CHO-Cav2.2GeneCACNA1BSourceshumanExpression systemCHOMethodwhole cell patch clampReference compoundNifedipine, verapamil, Cadmium (IC50=7.0µm±704nM)TargetPain, Spinal cord injury, Kleefstra syndrome 3、T-type calcium channel (CaV3.2) ChannelCaV3.2, T-typeCatalog Ref.ICE-CHO-Cav3.2GeneCACNA1HSourceshumanExpression systemHEK293Methodwhole cell patch clampReference compoundNifedipine, verapamil, NiClTargetConvulsion 4、P/Q-type calcium channel (CaV2.1)ChannelCav2.1Catalog Ref.ICE-CHO-Cav2.1GeneCACNA1ASourceshumanExpression systemCHOMethodwhole cell patch clampReference inhibitorCadmium (2.9 ± 0.658µM)Targetmigraine, seizure and ataxia syndromes