摘要
目的 对心肌细胞标记分子和与起搏活动相关的人超极化激活环核苷酸门控阳离子通道4（hHCN4）的表达进行检测,并进行细胞电生理学研究.方法 将20只成年新西兰大白兔分为对照组（10只）和实验组（10只）,对照组经心外膜注射腺病毒至左心室心尖部,实验组则注射hHCN4重组腺病毒.术后3d内每天描记两组大白兔体表心电图,术后第7天分离颈部双侧迷走神经干并进行双侧或单侧电刺激,以标测电极在心尖部进行起搏（刺激脉宽2～4 ms,刺激电压5～8V,刺激频率20 ～ 30次/s）,并记录同步肢体6导联心电图.免疫荧光检测两组大白兔心脏组织和实验组大白兔肝、肾、肺组织的hHCN4表达.膜片钳记录实验组大白兔基因转染后心尖部心肌单细胞动作电位起搏相关离子流If和LCa.结果 术后3d内心电图都没有记录到两组大白兔的室性心律失常事件.在术后第7天进行颈部迷走神经干电刺激时,实验组大白兔室性逸搏节律的频率明显快于对照组[（60±8）次/min比（42±6）次/min,P＜0.05],其室性逸搏节律起源于注射部位附近.实验组大白兔注射局部的心脏组织较对照组hHCN4蛋白表达明显增强,其肝、肾、肺组织未检测到该蛋白的表达.膜片钳记录到实验组大白兔基因转染后心尖部心肌单细胞存在起搏电流If和LCa.结论 hHCN4导入心肌可以使通道蛋白在局部过度表达,提高室性异位起搏点的频率.
Objective To detect the expression of myocardial cell marker and pacing activities-related human hyperpolarization-activated cyclic nucleotide-gated cation 4 （hHCN4）,and conduct cell electrophysiology study.Methods Twenty New Zealand white adult rabbits were divided into control group （10） and experimental group（10）.Attentive adventitial injection of adenovirus was injected to the left ventricular apex in rabbits of control group and hHCN4 recombinant adenovirus was injected to rabbits in experimental group respectively.Surface ECG of two groups was traced daily during 3 days postoperation.After 7 days,neck bilateral vagus nerves of rabbits were seperated and stimulated with bilateral or unilateral electrical stimulation.Pacing was established in apical area by mapping electrode （stimulation pulse width was 2-4 ms,stimulating voltage was 5-8 V,stimulation frequency was 20-30/s）,and synchronization limb 6-lead ECG was recorded.hHCN4 expression was detected in tissues of heart of rabbits from both groups and tissues of liver,kidney and lung of rabbits from experimental group by immunofluorescence method.If and LCa associated with action potential pacemaker ion flow of single apical myocardial cell was recorded by patch clamp.Results There were no records of ECG ventricular arrhythmia events during postoperative 3 days.When 7 days after electrical stimulation on vagus nerve,frequency of ventricular verapamil escape beats rhythm of the experimental group was significantly faster than that control group [（60±8）/min vs （42±6）/min,P＜0.05].Ventricular verapamil escape beats rhythm originated in the vicinity of the injection site.Overexpression of hHCN4 protein was detected in heart tissue of experimental group rabbits by immunofluorescence assay,while there were no detection of this protein in tissues of liver,kidney and lung in rabbits from experimental group.It was also confirmed that If and LCa existed in action potential pacemaker ion flow of single apical myocardial cell by patch clamp.Conclusions Transduction of hHCN4 into myocardial can lead to overexpression of channel protein in local area and increasement of the frequency of ventricular ectopic pacemaker.
出处
《中华生物医学工程杂志》
CAS
2014年第1期34-38,共5页
Chinese Journal of Biomedical Engineering
基金
河南省医学科技攻关计划项目（201203041、2011020012、201303068）
河南省教育厅科学技术研究重点项目科技攻关计划（13A320443）
河南省教育厅自然科学研究资助计划项目（2011A320028）
浙江省医药卫生科技计划项目（2011KYB164）
郑州大学第一附属医院青年基金（20110901）
舟山市科技计划项目基金（2011C12045）
志谢 本课题在设计、实施、汇总过程中,得到了相关同仁参与支持,在此一并署名致谢.北京大学人民医院心脏中心（李学斌、王龙、王伟民、刘健、段江波、苑翠珍、刘文玲、李翠兰、李蕾）
中国医学科学院北京协和医学院阜外心血管病医院心血管内科（庄晓峰）
郑州大学第一附属医院心血管内科（刘瑞云、魏经汉、黄振文、赵洛沙、张金盈、袁园、吕培生）,超声科（滑少华）
北京大学航天临床医学院（赵运涛）
成都市第三人民医院心血管内科（田芸）
上海交通大学附属仁济医院心血管内科（高谊）
首都医科大学附属北京安贞医院心血管内科12病房（韩红亚、杨世伟）
江门市中心医院心血管内科（任强）
舟山医院心血管内科（袁国裕、陈士良）
上海交通大学附属上海市第九人民医院心血管内科（薛超）
四川省人民医院心血管内科（陈丽竹）
重庆医科大学附属第一医院心血管内科（杨渊、王长鹰、王力、陆凯）
