【摘要】 目的:制备和纯化抗钠-钙交换体(Na⁺-Ca²⁺ exchanger,NCX)α-2_{（840-877）}单克隆抗体,并观察该抗体对大鼠缺血性心律失常的影响。方法:利用化学合成的NCXα-2_{（840-877）}肽段为免疫原免疫BALB/c小鼠,利用杂交瘤技术和间接ELISA法筛选,获得可稳定分泌NCXα-2_{（840-877）}单克隆抗体的杂交瘤细胞株。通过ELISA和SDS-PAGE等方法检测其效价和纯度。结扎冠脉左前降支建立大鼠缺血性心律失常模型。缺血前5 min用3F10（10μg/ml）抗体灌注离体心脏,在体实验经大鼠尾静脉注射3F10（50μg/kg）抗体,观察抗体大鼠室早数目、室速和室颤发生率和持续时间的影响。应用全细胞膜片钳技术观察3F10（10μg/ml）抗体对正常大鼠心肌细胞I_Na/Ca的影响。结果:①成功制备并获得抗NCXα-2_{（840-877）}单克隆抗体3F10,其效价和浓度分别为1∶40 960、1.62 mg/ml。②3F10抗体预处理可明显抑制大鼠离体和在体缺血性心律失常的发生。缺血前5 min给予3F10（10μg/ml）抗体预处理后,离体心脏的室速、室颤的发生率分别由100%和77.78%降至25%和25%,其持续时间和室早的个数也明显减少（P<0.05）。在麻醉大鼠,当给予3F10（50μg/kg）抗体预处理后,与模型组相比,大鼠室速和室颤的发生率分别由100%和83.33%降至33.33%和33.33%,其持续时间及室早的数目均明显减少（P<0.05）。③膜片钳实验发现3F10抗体（10μg/ml）对I_Na/Ca具有显著抑制作用。在钳制电位为+50 mV和-100 mV时,外向和内向I_Na/Ca分别由（3.23±0.05）和（-50.57±0.13）pA/pF降至（2.14±0.03）和（-3.14±0.06）pA/pF（P<0.05）。结论:成功获得一株可稳定分泌抗NCXα-2_{（840-877）}单克隆抗体的杂交瘤细胞株（3F10）,该抗体预处理对大鼠缺血性心律失常具有显著抑制作用,推测其作用机制与抑制心肌I_Na/Ca有关。更多还原

【Abstract】 Objective:To acquire monoclonal antibodies against α-2_{（840-877）} of Na⁺-Ca²⁺ exchanger,and to investigate the effects of the monoclonal antibody on cardiac arrhythmia in rats.Methods:BALB/c mice were immunized with a synthetic α-2_{（840-877）} peptide,and a hybridized tumor cell line capable of stable secretion of NCX α-2_{（840-877）} monoclonal antibody was obtained by hybridization and indirect ELISA.Its titer and purity were detected by ELISA and SDS-PAGE.The left anterior descending coronary artery was ligated to establish the ischemic arrhythmia model in vitro and in vivo in rats.Isolated heart was perfused with 10 μg/ml antibody 3 F10 5 min before ischemia,and for in vivo study,50 μg/kg antibody 3 F10 was applied through caudal vein.The effects of the antibody on number of premature ventricular beats,incidence and duration of ventricular tachycardia and ventricular fibrillation were observed in vitro and in vivo.Whole-cell patch clamp technique was used to record the effects of 10 μg/ml antibody on I_Na/Ca in rat ventricular myocytes.Results:①Monoclonal antibody 3 F10 against NCX α-2_{（840-877）}was obtained with its titer and concentration of 1∶40 960 and 1.62 mg/ml.②Antibody 3 F10 pretreatment significantly inhibited the occurrence of ischemic arrhythmias in vitro and in vivo in rats.After pretreatment with 3 F10 antibody,the incidence of ventricular tachycardia and ventricular fibrillation in isolated hearts decreased from 100% and 77.78% to 25% and 25% respectively,and the duration and number of premature ventricular beats decreased significantly（P<0.05）.Additionally,after 3 F10 antibody pretreatment in anaesthetized rats,the incidence of ventricular tachycardia and ventricular fibrillation decreased from 100% and 83.33% to 33.33% and 33.33% respectively,and the duration and number of premature ventricular beats significantly decreased（P<0.05）.③Electrophysiological study showed outward I_Na/Ca decreased from（3.23±0.05） to（2.14±0.03） pA/pF at holding potential of +50 mV（P<0.05）,and inward current decreased from（-5.57±0.13） to（-3.14±0.06） pA/pF at holding potential of-100 mV（P<0.05）.Conclusion:A hybridoma cell line stably secreting monoclonal antibody 3 F10 against NCX α-2_{（840-877）} is successfully obtained.Moreover,this antibody showed significant inhibitory effect on ischemic arrhythmias in rats,with the mechanism relating to inhibition of cardiac I_Na/Ca.更多还原