CAJ | 학술논문

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.
목적 대심기세포표기분자화여기박활동상관적인초겁화격활배핵감산문공양리자통도4(hHCN4)적표체진행검측,병진행세포전생이학연구.방법 장20지성년신서란대백토분위대조조(10지)화실험조(10지),대조조경심외막주사선병독지좌심실심첨부,실험조칙주사hHCN4중조선병독.술후3d내매천묘기량조대백토체표심전도,술후제7천분리경부쌍측미주신경간병진행쌍측혹단측전자격,이표측전겁재심첨부진행기박(자격맥관2～4 ms,자격전압5～8V,자격빈솔20 ～ 30차/s),병기록동보지체6도련심전도.면역형광검측량조대백토심장조직화실험조대백토간、신、폐조직적hHCN4표체.막편겸기록실험조대백토기인전염후심첨부심기단세포동작전위기박상관리자류If화LCa.결과 술후3d내심전도도몰유기록도량조대백토적실성심률실상사건.재술후제7천진행경부미주신경간전자격시,실험조대백토실성일박절률적빈솔명현쾌우대조조[(60±8)차/min비(42±6)차/min,P＜0.05],기실성일박절률기원우주사부위부근.실험조대백토주사국부적심장조직교대조조hHCN4단백표체명현증강,기간、신、폐조직미검측도해단백적표체.막편겸기록도실험조대백토기인전염후심첨부심기단세포존재기박전류If화LCa.결론 hHCN4도입심기가이사통도단백재국부과도표체,제고실성이위기박점적빈솔.
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.