NCX-2D2抗体对哇巴因诱发正常及心力衰竭大鼠心律失常的抑制作用及其机制研究

发布时间：2018-04-20 02:25

本文选题：全细胞膜片钳 + 钠-钙交换体　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:建立哇巴因诱发正常和心力衰竭大鼠心律失常模型,观察抗钠-钙交换体NCX-2D2抗体对模型大鼠心律失常的影响,并分析其可能的作用机制。方法:1.哇巴因诱发成年大鼠离体和在体心脏心律失常模型的建立(1)哇巴因诱发大鼠离体心律失常模型健康SD大鼠,50 mg·kg~(-1)戊巴比妥钠腹腔注射麻醉后,开胸游离心脏并且悬挂于Langendorff装置上,进行主动脉逆行灌流,流速为8mL·min-1,观察至少30 min待心电图波形稳定后使用给药泵给药,记录心电图。实验分组如下:①正常对照组:含2.5 mmol·L-1 CaCl2的台氏液灌流;② 2D2+CaCl2 组:含 10 μg·ml~(-1) NCX-2D2 抗体和 2.5 mmol·L-1 CaCl2 的台氏液灌流;③ Oua+CaCl2组:含5μmol·L-1 Ouabain 和 2.5 mmol·L-1 CaCl2 的台氏液灌流;④ 2D2+Oua+CaCl2 组:含 10 μg ml~(-1) NCX-2D2 抗体、5 μmol·L-1 Ouabain 和 2.5 mmol.L-1 CaCl2的台氏液灌流。心电图记录药物干预后30 min内室性期前收缩个数,室速和室颤发生率及持续时间。(2)哇巴因诱发麻醉大鼠心律失常模型健康SD大鼠用30 mg·kg~(-1) 水合氯醛腹腔注射麻醉后将大鼠置于鼠板上,连接固定肢体导联。采用BL-420F生物机能实验系统记录大鼠Ⅱ导联心电图,连续观察30min心电图无异常后尾静脉给药,记录药物干预后1 h内室性期前收缩的个数、室速和室颤持续时间及发生率。2.大鼠心力衰竭模型的制备取成年健康SD大鼠,随机分为伪手术组和心衰组,术前禁饮禁食24 h,称量大鼠体重,给予水合氯醛(30 mg·kg~(-1))腹腔注射麻醉。待大鼠麻醉后固定于鼠板上,脱去大鼠腹部的毛,于大鼠左脊肋角后纵向切口 2 cm,钝性分离暴露腹主动脉,以7号针针头与腹主动脉一起结扎,然后抽出针头形成腹主动脉狭窄,关闭腹腔。伪手术组仅不结扎腹主动脉,其余操作均与心衰组相同。术后3天每天腹腔注射青霉素20万单位预防感染。3.超声心动图检测及HE染色于造模后12周进行检测。超声心动图检测以下指标:左室舒张末期内径(LVIDd)、左室收缩末期内径(LVIDs)、左室射血分数(LVEF)和左室短轴缩短率(LVFS)。HE染色观察伪手术组和心衰组大鼠心肌组织形态学特点。心功能检测完毕的大鼠,开胸取心脏,置于提前预冷的4℃生理盐水中,经主动脉灌流冲洗干净残存血液后,分离心房心室,浸泡于4%多聚甲醛固定液中,固定24小时后常规脱水,石蜡包埋,心室肌组织切片,厚度为5μm,经HE染色,然后镜下观察。4.哇巴因诱发心力衰竭大鼠并发心律失常模型及心功能测定取心力衰竭SD大鼠用水合氯醛(30 mg·kg~(-1))腹腔注射麻醉后,仰卧位固定于鼠板上,连接肢体导联,记录大鼠Ⅱ导联心电图;同时通过右颈总动脉插管至左心室腔测定心功能,并经由颈总动脉心室插管给药的方式给予哇巴因(0.4 mg·kg~(-1))制备心力衰竭大鼠并发急性心律失常模型,NCX-2D2抗体(40、60μg·kg~(-1))、胺碘酮(Amiodarone,3mg-kg~(-1))分别于哇巴因注射前5分钟经颈总动脉插管给予,观察给药后1h期前收缩次数、室速和室颤发生率、持续时间的变化;并同步记录心率、左室最大收缩速率、左室最大舒张速率、左室舒张压。5.大鼠心室肌细胞急性分离取健康SD大鼠,50mg·kg~(-1)戊巴比妥钠腹腔注射麻醉后,颈动脉放血后,快速开胸取心脏,修剪后经主动脉逆行插管悬挂于Langendorff灌流装置上,以无钙且100%氧气的台氏液灌流心脏8～10分钟,灌注环境:恒压(75 cmH2O)、恒温(37℃),后改为胶原酶液(0.07-0.1 g·L-1)循环灌流15～20分钟,观测心脏肌组织变大、变软,冠脉血管边缘不清时迅速剪下左心室,用KB液快速冲洗后置于其中,眼科剪剪至2～3mm3小块,用玻璃吸管(尖端圆润,以免损伤肌细胞)轻轻吹打3～5 min后即可。过滤,弃去残留心脏组织,滤液置于KB液中保存,后将其置于常温稳定3小时后使用。6.全细胞膜片钳记录应用全细胞膜片钳技术,在电压钳模式下记录大鼠左心室肌细胞L-型钙电流(ICa-L),Na+/Ca2+交换电流(INa/Ca);在电流钳模式下记录大鼠左心室肌细胞动作电位(AP),施加条件刺激(15 ms 3Hz)诱发延迟后除极。结果:1.NCX-2D2抗体对哇巴因诱发大鼠心律失常具有显著抑制作用(1)10 μg·ml~(-1) NCX-2D2抗体可明显抑制哇巴因诱发大鼠离体心脏心律失常的发生,在哇巴因组,在给药30min内100%大鼠发生室速,71.43%大鼠出现室颤,在给予10μg·ml~(-1) NCX-2D2抗体干预后,在观察期(30min)内大鼠室速和室颤发生率分别降低至28.5%和0,室速持续时间由(192.71±18.45)s缩短至(11.85±20.27)s(P0.05),室性期前收缩个数由303±26减少至78±7(P0.05)。(2)NCX-2D2抗体可明显抑制哇巴因诱发麻醉大鼠心律失常的发生。在哇巴因组,在给药后1小时内100%大鼠发生室速,62.5%大鼠出现室颤。在预先5分钟给予60、80μg·kg~(-1)NCX-2D2抗体干预后,在观察期(1h)内大鼠室速发生率分别降低为50%、37.5%,室速持续时间由(50.91±3.58)min分别缩短至(4.29±4.78)、(0.14±0.21)min(P0.05),大鼠室颤发生率分别降低为33.3%、0,60μg·kg~(-1) NCX-2D2 抗体使室颤持续时间由(2.83±3.03)min 缩短至(0.37±0.58)min(P0.05)。经比较发现,80 μg·kg~(-1)NCX-2D2抗体对哇巴因诱发心律失常的抑制作用与阳性对照组胺碘酮效果相似。2.腹主动脉缩窄术后12周,大鼠心力衰竭模型制备成功由M型超声心动图及HE染色发现,大鼠腹主动脉缩窄术后12周,大鼠出现心力衰竭。M型超声心动图显示,大鼠左室收缩末期内径和左室舒张末期内径均明显增大,左室收缩末期内径由(2.05±0.08)mm增大至(5.42±0.29)mm(P0.05),左室舒张末期内径由(5.25±0.19)mm增大至(7.59±0.36)mm(P0.05);左室射血分数由(92±1.37)%减小至(62±5.65)%(P0.05),左室短轴缩短率由(58±2.48)%减小至(30±2.85)%(P0.05)。HE染色显示,心力衰竭大鼠细胞排列紊乱,心肌细胞出现肥大,部分心肌组织出现纤维化。3.NCX-2D2抗体对哇巴因诱发心力衰竭大鼠并发的心律失常具有显著抑制作用研究结果显示,0.4 mg·kg~(-1)哇巴因可明显增强心力衰竭大鼠心功能,但同时诱发出严重的心律失常。预先给予40、60μg·kg~(-1) NCX-2D2抗体干预后,哇巴因诱发心衰大鼠并发的心律失常被明显抑制,且大鼠心功能较心衰组明显提高。在哇巴因组100%大鼠发生室速,66.7%大鼠发生室颤,在给予哇巴因前5 min给予40、60μg·kg~(-1) NCX-2D2抗体干预后,在观察期(1h)内室速发生率分别降低至66.7%和33.3%,在给予哇巴因前5 min给予40、60μg·kg~(-1) NCX-2D2抗体干预后,室速发生率分别降低至66.7%和33.3%,室速持续时间由(1120.8±173.9)s分别缩短至(264.6±206.4)、(8.6±13.5)s(P0.05),室颤发生率分别降低至 33.3%和 16.7%,室颤持续时间由(22.50±24.31)s 分别缩短至(3.70±6.10)、(1.50±3.67)s(P0.05),室性期前收缩个数由1640±157分别降低至488±19和138±33(P0.05)。经比较发现,60μg·kg~(-1) NCX-2D2抗体对室性期前收缩的抑制作用较阳性对照组胺碘酮明显,对其他类型心律失常的抑制作用与阳性对照组胺碘酮相似,且其心功能较心衰组大鼠明显提高。4.NCX-2D2抗体可部分逆转哇巴因对大鼠单个心室肌细胞钠-钙交换电流(INa/Ca)的增大作用在钳制电位+50mV和-100mV时,哇巴因组心肌细胞INa/Ca分别为9.20±0.09和-11.42±0.27(pA/pF),与对照组 4.25±0.05 和-4.08±0.09(pA/pF)相比明显增大(P0.05)。在哇巴因存在的情况下联合给予5μg·ml~(-1)NCX-2D2抗体后,INa/Ca在+50mV和-100mV时的外向和内向电流分别降低至6.67±0.27和-7.42±0.26(pA/pF),均明显低于哇巴因组(P0.05),但仍未完全恢复至正常水平(P0.05)。5.NCX-2D2抗体可部分逆转哇巴因对大鼠单个心室肌细胞L-型钙电流(ICa-L)的增大作用研究结果显示,哇巴因组心肌细胞ICa-L为-8.73±0.36(pA/pF),与对照组-6.30±0.53(pA/pF)相比明显增大(P0.05)。在联合应用5μg·ml~(-1) NCX-2D2抗体后,ICa-L降至-7.45±0.29(pA/pF),与哇巴因组相比明显降低(P0.05),但仍未完全恢复至正常水平(P0.05)。6.NCX-2D2抗体可显著抑制哇巴因诱发的大鼠单个心室肌细胞延迟后除极研究结果显示,对照组和2D2抗体组均未出现延迟后除极(DADs)。哇巴因组大鼠单个心室肌细胞DADs发生率为83.33%,在给予5μg·ml~(-1)NCX-2D2抗体干预后,大鼠单个心室肌细胞DADs发生率降低为8.3%。与哇巴因组相比,抗体干预组(哇巴因+2D2抗体组)大鼠单个心室肌细胞DADs发生率明显降低(P0.05)。结论:NCX-2D2抗体可显著抑制哇巴因诱发正常及心力衰竭大鼠并发的心律失常,其抗心律失常机制主要与抑制钠-钙交换和L-型钙通道以减轻心肌细胞钙超载和抑制心肌细胞延迟后除极有关。
[Abstract]:Objective: to establish an arrhythmia model of normal and heart failure rats induced by ouabain, observe the effect of anti sodium calcium exchange NCX-2D2 antibody on the arrhythmia of model rats, and analyze its possible mechanism. Methods: 1. ouabain induced adult rat in vitro and in vivo cardiac arrhythmia model (1) ouabain induced rat in vitro Cardiac arrhythmia model healthy SD rats, 50 mg. Kg~ (-1) pentobarbital sodium intraperitoneal injection anaesthesia, open the chest free heart and hang on the Langendorff device, the aorta retrograde perfusion, the flow rate of 8mL min-1, observe at least 30 min after the electrocardiogram wave stability after the use of drug pump administration, record electrocardiogram. Experimental groups are as follows: 1 normal group: 1 normal Control group: the irrigation of table's solution containing 2.5 mmol / L-1 CaCl2; (2) 2D2+CaCl2 group: 10 micron g / ml~ (-1) NCX-2D2 antibody and 2.5 mmol L-1 CaCl2 liquid perfusion. Uabain and 2.5 mmol.L-1 CaCl2's liquid perfusion. Electrocardiogram recorded the number of ventricular premature contractions, ventricular tachycardia and ventricular fibrillation in 30 min after drug intervention. (2) ouabain induced arrhythmia model healthy SD rats were placed on the rat board with 30 mg kg~ (-1) chloral chloral abdominal cavity injection. The BL-420F biological function test system was used to record the electrocardiogram of rat II lead, and the 30min electrocardiogram was continuously observed without abnormal tail vein. The number of ventricular premature contraction in 1 h after drug intervention, the ventricular tachycardia and the duration of ventricular fibrillation and the heart failure model of.2. rats were prepared for the preparation of adult healthy SD rats. The rats were divided into the puppet group and the heart failure group, 24 h before the operation, the weight of the rats were weighed and the chloral hydrate (30 mg. Kg~ (-1)) was given intraperitoneal injection. After the rats were anesthetized, the rats were fixed on the rat plate and removed the hair of the rat abdomen. The longitudinal incision after the left rib angle of the rat was 2 cm, the abdominal aorta was exposed by blunt separation, and the needle head of the 7 needle and the abdominal aorta were separated. Ligation, and then draw out the needle to form the abdominal aorta stenosis and close the abdominal cavity. The pseudo operation group only did not ligate the abdominal aorta, and the rest of the operation were the same as the heart failure group. 3 days after the operation, the celiac injection of penicillin 200 thousand units to prevent infection.3. echocardiography and HE staining at 12 weeks after the operation. The following index of the echocardiography: left ventricle: left ventricle The end diastolic diameter (LVIDd), the left ventricular end systolic diameter (LVIDs), the left ventricular ejection fraction (LVEF) and the short axis shortening rate (LVFS).HE staining were used to observe the histomorphological characteristics of the myocardium of the rats in the PN group and the heart failure group. The rats after the cardiac function test were taken into the heart by opening the chest, and placed in the pre cooled 4 centigrade normal saline, and irrigated through the aorta. After a clean and residual blood, the atrial ventricle was separated and soaked in 4% polyformaldehyde fixed solution. After 24 hours fixed dehydration, paraffin embedded and ventricular muscle tissue section, the thickness was 5 mu m, and HE staining was used to observe the arrhythmia model of heart failure induced by.4. ouabain and the cardiac function determination of heart failure SD rats with chlorinated chloride. After intraperitoneal injection of aldehyde (30 mg. Kg~ (-1)), the supine position was fixed on the rat plate, connected to the limb lead, and recorded the electrocardiogram of the rat II lead. At the same time, the cardiac function was measured by the right common carotid artery intubation to the left ventricle and ouabain (0.4 mg. Kg~ (-1)) was given by the way of the ventricular catheterization of the common carotid artery to prepare the acute heart failure rats. The arrhythmia model, NCX-2D2 antibody (40,60 g / kg~ (-1)) and amiodarone (Amiodarone, 3mg-kg~ (-1)) were given respectively by the common carotid artery intubation 5 minutes before ouabain injection. The number of pre systolic times, the rate of ventricular tachycardia and ventricular fibrillation, the duration of duration were observed, and the heart rate, the maximum systolic velocity of left ventricle and the maximum left ventricular diastolic speed were recorded simultaneously. Rate, left ventricular diastolic pressure.5. rat ventricular myocytes were isolated from healthy SD rats. After intraperitoneal injection of 50mg. Kg~ (-1) pentobarbital sodium, the heart was quickly opened after the carotid artery bleed. After pruning, the aortic retrograde intubation was suspended on the Langendorff perfusion device and perfusion of the heart without calcium and 100% oxygen for 8~10 minutes. Environment: constant pressure (75 cmH2O), constant temperature (37 degrees C), then converted to collagenase fluid (0.07-0.1 G. L-1) for 15~20 minutes. The observation of cardiac muscle tissue becomes larger and softer. The left ventricle is quickly cut off when the coronary artery is not clear. After rapid flushing of the KB solution, the eye section is cut to 2 to 3mm3 small pieces, and the glass tube is rounded to avoid injury of muscle fine. After blowing 3~5 min gently, the cells were filtered and abandoned to the residual cardiac tissue, the filtrate was stored in the KB solution, and then the whole cell patch clamp technique was used to record the whole cell patch clamp technique using the whole cell patch clamp of.6. after 3 hours at normal temperature, and the L- type calcium current (ICa-L) and Na+/Ca2+ exchange current (INa/Ca) were recorded in the rat left ventricular myocytes under the voltage clamp mode. The action potential (AP) of rat left ventricular myocytes (AP) was recorded under current clamp mode, and conditioned stimulation (15 ms 3Hz) induced depolarization. Results: 1.NCX-2D2 antibody had significant inhibitory effect on ouabain induced arrhythmia induced by ouabain (1) 10 mu g. Ml~ (-1) NCX-2D2 antibody could obviously inhibit the hair cardiac arrhythmia induced by ouabain. In ouabain group, 100% rats in ouabain group had ventricular tachycardia and 71.43% rats had ventricular fibrillation. The rate of ventricular tachycardia and ventricular fibrillation was reduced to 28.5% and 0 in the observation period (30min). The duration of ventricular tachycardia was shortened from (192.71 + 18.45) s to (11.85 + 20.27) s (P0.05) and ventricular premature contraction in the observation period (30min). The rate of ventricular tachycardia was reduced to 28.5% and 0 in the observation period (30min). The number decreased from 303 + 26 to 78 + 7 (P0.05). (2) NCX-2D2 antibody could obviously inhibit the occurrence of arrhythmia in ouabain induced rats. In ouabain group, the ventricular tachycardia occurred in 100% rats in 1 hours after administration, and ventricular fibrillation in 62.5% rats. 60,80 uh G. Kg~ (-1) NCX-2D2 antibody was given in advance for 5 minutes, and the rat ventricular tachycardia occurred in the observation period (1H). The rate was reduced to 50%, 37.5%, and the duration of room speed was shortened from (50.91 + 3.58) min to (4.29 + 4.78), (0.14 + 0.21) min (P0.05), and the incidence of ventricular fibrillation in rats was reduced to 33.3%, and 0,60 mu g. Kg~ (-1) NCX-2D2 antibody shortened the duration of ventricular fibrillation from (2.83 + 3.03) min to (0.37 + 0.58) min (P0.05). The inhibitory effect of antibody on ouabain induced arrhythmia was similar to that of positive control histamine iodide. 12 weeks after.2. abdominal aorta coarctation, the rat model of heart failure was successfully prepared by M echocardiography and HE staining, 12 weeks after the abdominal aorta coarctation in rats. The rats had heart failure.M echocardiography, and the left ventricle of rats The end systolic diameter and left ventricular end diastolic diameter increased significantly. The end-stage internal diameter of left ventricular systole increased from (2.05 + 0.08) mm to (5.42 + 0.29) mm (P0.05). The end diastolic diameter of left ventricle increased from (5.25 + 0.19) mm to (7.59 + 0.36) mm (P0.05), and left ventricular ejection fraction decreased from (92 + 1.37)% to (62 5.65)% (P0.05), and the short axis shortening rate of left ventricle was (58 + 5.25). % to (30 + 2.85)% (P0.05)% (P0.05).HE staining showed that the cells in the heart failure rats were arranged in disorder, the cardiac myocytes appeared hypertrophy, and some myocardial tissue appeared fibrosis.3.NCX-2D2 antibody. The results showed that 0.4 mg. Kg~ (-1) wow Ba could obviously enhance the heart of heart failure induced arrhythmia in rats. The heart function of the rats was induced by force, but the cardiac arrhythmia was induced at the same time. The prognosis of 40,60 mu g. Kg~ (-1) NCX-2D2 antibody was given in advance. The arrhythmia in the rats induced by ouabain induced heart failure was obviously inhibited, and the heart function of the rats was significantly higher than that in the heart failure group. In ouabain group, 100% rats had ventricular tachycardia, and the 66.7% rats had ventricular fibrillation and were given wow. The prognosis of 40,60 mu g. Kg~ (-1) NCX-2D2 antibody was given to the first 5 min of Ba, and the rate of ventricular tachycardia in the observation period (1H) decreased to 66.7% and 33.3% respectively. The rate of ventricular tachycardia decreased to 66.7% and 33.3%, respectively, and the duration of ventricular tachycardia was reduced to (264.6. 173.9) to 264.6 min before giving ouabain. (8.6 + 13.5) s (P0.05), the incidence of ventricular fibrillation was reduced to 33.3% and 16.7% respectively. The duration of ventricular fibrillation was shortened from (22.50 + 24.31) s to (3.70 + 6.10), (1.50 + 3.67) s (P0.05). The number of ventricular premature contraction decreased from 1640 + 157 to 22.50 + 24.31 and P0.05. The inhibitory effect of contraction was more obvious than that of the positive control histamine iodide, and the inhibitory effect on other types of arrhythmia was similar to that of the positive control histamine iodide, and the heart function was significantly higher than that of the heart failure group. The.4.NCX-2D2 antibody could partly reverse the effect of ouabain on the increase of sodium calcium exchange current (INa/Ca) in single ventricular myocytes of rats. The INa/Ca of cardiac myocytes in ouabain group was 9.20 + 0.09 and -11.42 + 0.27 (pA/pF), respectively, compared with 4.25 + 0.05 and -4.08 + 0.09 (pA/pF) in the control group (P0.05). Under the presence of ouabain 5 u g ml~ (-1) NCX-2D2 antibodies, the extroverted and introverted currents were reduced respectively. 6.67 + 0.27 and -7.42 + 0.26 (pA/pF) were significantly lower than ouabain group (P0.05), but still not completely recovered to normal level (P0.05).5.NCX-2D2 antibody could partly reverse the effect of ouabain on the increase of L- type calcium current (ICa-L) in rat single ventricular myocytes. The myocardial ICa-L in ouabain group was -8.73 + 0.36 (pA/pF), and control Group -6.30 + 0.53 (pA/pF) was significantly increased (P0.05). After combined use of 5 g. Ml~ (-1) NCX-2D2 antibody, ICa-L decreased to -7.45 + 0.29 (pA/pF), compared with ouabain group (P0.05), but still not completely recovered to normal level (P0.05) could significantly inhibit the depolarization of rat single ventricular myocytes induced by ouabain. The results showed that there was no delayed depolarization (DADs) in the control group and the 2D2 antibody group. The incidence of DADs in single ventricular myocytes in ouabain group was 83.33%. The incidence of DADs in single ventricular myocytes in rats was reduced to 8.3%. compared to ouabain group, and the antibody intervention group (ouabain +2D2 antibody group) was compared with 5 g. Ml~ (-1) NCX-2D2 antibody. The incidence of DADs in rat single ventricular myocytes was significantly decreased (P0.05). Conclusion: NCX-2D2 antibody can significantly inhibit the arrhythmia induced by ouabain and normal and heart failure rats. Its antiarrhythmic mechanism is mainly related to the inhibition of sodium calcium exchange and L- type calcium channel to reduce myocardial cell calcium overload and inhibit myocardial cell delay. Close.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541

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