胚胎心肌细胞移植治疗缓慢型心律失常的实验研究

目的探讨胚胎心肌细胞移植重建心脏优势起搏点治疗缓慢型心律失常的可行性.方法酶法分离引产男性胎儿心房组织(包括窦房结),获取单个心肌细胞,差速贴壁法纯化,4、6-二脒基-2-苯基吲哚(DAPl)标记后制备成5×106个/ml心肌细胞悬液,开胸直视下将1ml悬液注入Yorkshire猪左室游离壁(n=2),对照组(n=2)注入等体积培养基DMEM,移植前3天开始应用环孢素A(Ⅳ,5mg/kg/d)和泼尼松龙(Ⅳ,2.5mg/kg/d)抑制免疫排斥反应.移植后2～3周应用射频消融技术打断希氏束建立完全性房室传导阻滞动物模型,分别应用心电图、Holter、免疫荧光显像、心腔内起搏标测和激动标测进行组织学和电生理学评价,RT-PCR检测各组Y染色体SRYmRNA表达情况.结果希氏束消融后,两组动物均形成完全性房室传导阻滞,细胞移植组室性心律频率为75±30bpm,对照组为室性逸搏心律,频率为(35±10)次/min或交界心律,最后发生室颤死亡.心腔内起搏标测和激动标测证实细胞移植组室性心律起源于细胞注射表达.心脏组织冰冻切片可见DAPI标记的呈蓝色荧光的移植细胞核,移植细胞与宿主心肌细胞间有连结蛋白43(connexin43)和N型钙粘素(N-Cadherin)的表达,说明移植细胞存活并与宿主心肌细胞形成电一机械联系.RT-PCR检测到细胞移植组SRY基因片段区,对照组未扩增出SRY片段,说明胚胎心肌细胞在移植区存活.结论心肌细胞移植可能取代电子起搏器治疗缓慢型心律失常.     

自体骨髓间叶干细胞诱导分化移植治疗房室阻滞的初步观察

利用诱导分化的骨髓间叶干细胞自体移植治疗房室阻滞,探讨生物介入方法治疗缓慢型心律失常的新途径。11只实验犬随机分为实验组(n=6)和对照组 (n=5)。应用射频技术消融His束,制备永久Ⅲ度房室阻滞动物模型;每只犬抽取 10ml犬骨髓液,应用密度梯度离心法及贴壁培养法分离、培养和扩增骨髓间叶干细胞, 5 氮胞苷对骨髓间叶干细胞进行诱导分化;房室阻滞 4周后,开胸心脏直视下将BrdU标记的分化干细胞 (1ml, 1. 5×107细胞)多点注射至该犬消融的His束部位,而对照组仅将 1mlDMEM培养液替代干细胞悬液多点注射至相同部位。术后 1~12周,应用心电图观察两组活体动物房室功能恢复情况,应用组织病理、免疫组化等技术对移植干细胞的存活、增殖、分化与缝隙连接功能进行评价。结果:在动物房室阻滞模型中,实验组犬在自体骨髓干细胞移植后 12周, 2 /6只犬的房室传导功能得以改善;病理与免疫组化示诱导分化的骨髓干细胞在His束区存活、增殖和分化为心肌细胞、血管内皮细胞,并与宿主细胞建立缝隙连接;而对照组 5只犬未见上述变化。结论:自体移植诱导分化的骨髓间叶干细胞有可能改善His束传导功能。     

自体骨髓间叶干细胞诱导分化移植治疗房室阻滞的初步观察水

利用诱导分化的骨髓间叶干细胞自体移植治疗房室阻滞,探讨生物介入方法治疗缓慢型心律失常的新途径。11只实验犬随机分为实验组(n=6)和对照组 (n=5)。应用射频技术消融His束,制备永久Ⅲ度房室阻滞动物模型;每只犬抽取 10ml犬骨髓液,应用密度梯度离心法及贴壁培养法分离、培养和扩增骨髓间叶干细胞, 5 氮胞苷对骨髓间叶干细胞进行诱导分化;房室阻滞 4周后,开胸心脏直视下将BrdU标记的分化干细胞 (1ml, 1. 5×107细胞)多点注射至该犬消融的His束部位,而对照组仅将 1mlDMEM培养液替代干细胞悬液多点注射至相同部位。术后 1~12周,应用心电图观察两组活体动物房室功能恢复情况,应用组织病理、免疫组化等技术对移植干细胞的存活、增殖、分化与缝隙连接功能进行评价。结果:在动物房室阻滞模型中,实验组犬在自体骨髓干细胞移植后 12周, 2 /6只犬的房室传导功能得以改善;病理与免疫组化示诱导分化的骨髓干细胞在His束区存活、增殖和分化为心肌细胞、血管内皮细胞,并与宿主细胞建立缝隙连接;而对照组 5只犬未见上述变化。结论:自体移植诱导分化的骨髓间叶干细胞有可能改善His束传导功能。     

三维标测系统指导下房室结解剖消融及其对His束起搏参数的影响

目的 探讨三维标测系统指导下,对房室结解剖位置消融及其对His束起搏参数的影响.方法 入选房室结消融的永久性心房颤动患者28例,其中7例既往接受了His束起搏,仅行房室结消融(单一消融组),21例同时进行了三维标测系统指导下房室结消融联合His束起搏(联合手术组).三维标测系统下标测His束、冠状窦口及三尖瓣环,在构建...     

不同位点组合同步双室起搏对慢性心力衰竭心脏做功效率的影响

目的　探讨不同位点组合同步心室起搏模式对慢性心力衰竭心脏做功效率的影响。方法　 1 4只慢性心力衰竭猪模型被随机组合为右房 -近希氏束起搏组 (RA- c His B)、右房 -右室心尖 -左室后侧壁起搏组 (RA- RVA- LVPL)和右房 -近希氏束 -左室后侧壁起搏组 (RA- c His B- LVPL)。分别比较起搏后各组心排血量 (CO)和心脏做功效率 (CWE)。结果　 RA- c His B- LVPL三腔起搏的 CWE、CO分别较 RA- RVA- LVPL三腔起搏、RA-c His B房室顺序起搏显著提高 (P均 <0 .0 5) ,而 RA- RVA- LVPL三腔起搏和 RA- c His B房室顺序起搏之间 CWE、CO无显著性差异 (P>0 .0 5)。结论　 RA- c His B- LVPL三腔起搏的各项参数指标优于其它组合模式起搏 ,在保持房室激动顺序的同时 ,双室同步起搏更有利于提高心脏做功效率。     

鼠胚胎干细胞早期分化来源的心肌细胞同种异体移植后的电生理特性

目的 探讨小鼠胚胎干细胞早期分化来源的心肌细胞进行同种异体移植后的电生理特性.方法 首先通过电转染构建a-MHC-EGFP-ESc系[将心肌细胞特异的OL-MHC启动子与表达基因--绿色荧光蛋白(EGFP)基因融合,构建成真核表达载体],悬滴法诱导胚胎干细胞分化,在分化早期(7+4)d利用流式细胞仪筛选带绿色荧光的心肌细胞,将纯化的心肌细胞(5×106个/ml)移植到小鼠心室壁,对照组注入等体积培养基,移植前3 d开始应用环孢素A(静脉注射,5 mg·kg-1·d-1)和泼尼松龙(静脉注射,2.5 mg·kg-1·d-1)抑制免疫排斥反应.移植后2周分离两侧颈迷走神经行电刺激抑制窦房结与房室结,记录刺激前后的体表心电图,然后分别行免疫荧光显像和膜片钳研究.结果 刺激迷走神经前,移植组和对照组均呈正常的窦性心律,两组无室性心律失常的发生;刺激迷走神经后,两组动物均出现异位的心室起搏心律,细胞移植组与对照组心室频率无差异.移植区冰冻切片免疫荧光分析可见EGFP标记的移植细胞具有肌钙蛋白I(cTnI)表达,说明分化细胞移植后仍具有心肌特性,且移植细胞与宿主心肌细胞间有连接蛋白43的表达,表明移植细胞与宿主心肌细胞间形成电偶联通道.膜片钳分析,绿色荧光细胞移植前后其具有起搏细胞动作电位的比例分别为(85.1%vs 1 1.4%,P＜0.05),该类细胞在移植前后的起搏电流强度有所增强((11.2±2.4)pA/pF vs(15.5±1.9)pA/pF,P＜0.05].移植区分离的绿色荧光细胞中48%具有心室肌动作电位.结论 胚胎干细胞早期分化来源的心肌细胞在体移植后能进一步分化为成熟的心室肌细胞及起搏细胞.     

成熟心肌细胞异体移植对心肌梗死后心功能的保护作用

目的:研究异体成熟心肌细胞移植对心肌梗死后心功能的保护作用。方法:成年新西兰兔结扎冠状动脉左前降支建立心肌梗死模型,体外培养异体心肌细胞并用5溴脱氧尿核苷（BrdU）标记,将4×107/ml细胞悬液移植到急性心肌梗死区域。对照组将同量细胞培养液注射到急性心肌梗死区。细胞移植5周后超声心动图测量活体心功能,并进行组织学观察及免疫组化检测。结果:接受细胞移植组心脏功能显著优于对照组,左室重构受到抑制。细胞移植区观察到被标记心肌细胞,同时电镜下观察到心肌细胞之间存在细胞间联系。结论:移植细胞能够在宿主体内存活并与周围组织建立联系抑制左室重构,保护心脏功能。     

股动脉逆行法建立完全性房室传导阻滞动物模型

目的研究经股动脉逆行途径射频消融His束建立完全性房室传导阻滞(AVB)动物模型的可行性。方法 15～20kg健康雄性杂种犬8只,麻醉开胸后于左室缝制临时起搏电极及记录电极。在X线引导下,将消融电极经股动脉逆行至主动脉无冠窦,标测定位并射频消融His束。分别记录每次实验的操作和消融时间,比较消融前后的心率及心律变化,观察术后并发症情况。结果 8只犬成功建立完全性AVB动物模型,操作时间17.5(1,25.25)min,消融时间84(10,90)s。所有犬在消融前均为窦性心律,心率(168±13)次/分,消融时出现交界性心律,消融完成后体表及腔内心电图均显示完全性AVB,心率(57±11)次/分。结论通过股动脉逆行途径射频消融His束建立完全性房室传导阻滞动物模型的方法简便可行、成功率高。     

HCN4通道基因过度表达的电生理实验研究

目的探讨腺病毒介导超极化激活环核苷酸门控通道（HCN4）在猪左心室过度表达的电生理效应。方法应用细菌内同源重组法构建携带人HCN4基因的重组腺病毒载体（Ad-HCN4）和绿色荧光蛋白的重组腺病毒载体（Ad-GFP）。实验动物随机分成3组：Ad-HCN4组（n=6），Ad-GFP组（n=5）和PBS组（n=3）。腺病毒或PBS注入到Yorkshire猪左心室游离壁，3-4d后行房室结消融造成完全性房室阻滞，记录体表心电图并进行心内起搏标测。注射部位心肌组织用酶分离后获得单个心肌细胞，膜片钳记录起搏电流（If）和内向整流钾电流（Ik1）。结果Ad-HCN4组室性心律频率显著快于Ad-GFP组和PBS组，心腔内起搏标测证实该心律起源于Ad-HCN4注射区且可被异丙肾上腺素所调节，无水乙醇消融Ad-HCN4注射区后该心律消失。Ad-HCN4转染心肌细胞表达较大的If;Ik1在3组间差异无统计学意义。结论腺病毒介导HCN4通道基因心室局部高表达发挥了生物起搏器的作用。     

起搏治疗对心脏自主神经基质改变的实验研究

目的观察窦房结和房室结功能障碍对心脏神经基质的影响,以及右心耳（RAA）和右心室心尖部（RVA）起搏对神经重构的作用。方法28只犬随机分为窦房结损伤组、RAA起搏组、房室结损伤组和RVA起搏组。7只健康犬作为对照组。14只犬用20％甲醛滤纸片外敷窦房结区损伤窦房结,其中7只犬将起搏电极导线缝合固定于RAA上,以90~．／min行心房起搏。另外14只犬于房室交界区注入无水乙醇损伤房室结,其中7只犬将起搏电极导线缝合固定于RVA,以907~／min起搏心室。起搏60d后二次麻醉,取出心脏。所有犬均于RAA、房间隔（As）、左心耳（LAA）、RVA、室问隔（Vs）、左心室心尖部（LVA）取材。运用免疫组化技术测定心肌中的新生神经（GAP43标测）和交感神经（TH标测）密度。结果（1）窦房结损伤组RAA的新生神经和交感神经密度低于正常对照组（P〈0．01）,但AS、LAA及心室的新生神经和交感神经密度与对照组相似（P〉0．05）;（2）RAA起搏组RAA的新生神经与交感性神经密度高于窦房结损伤组（P〈0．01）,与对照组接近（P〉0．05）;（3）房室结损伤组心房和心室各部位的新生神经和交感神经密度高于对照组（P〈0．01）;（4）RVA起搏组心房和心室的新生神经和交感神经密度与房室结损伤组差异无统计学意义（P〉0．05）。结论窦房结和房室结功能障碍可造成心脏神经基质的改变。RAA起搏可以逆转窦房结功能低下造成的神经重构,而RVA起搏不能逆转。     

自体心耳肌细胞移植对心功能的影响

目的将急性分离的兔自体左心耳心肌细胞移植到自体梗死区心肌,以研究其对心功能的改善情况。方法结扎成年兔的冠状动脉前降支,建立心肌梗死模型,4周后获取自体左心耳组织,急性消化分离为单细胞后分别将细胞悬液和培养液注射到移植组和未移植组梗死区内。4周后行超声心动图检查。结果4周后移植组与未移植组兔子全部存活。超声心动图检查,至移植后第4周,移植组左室功能各项指标好于未移植组。结论急性分离的自体左心耳心肌细胞移植到梗死区心肌内能够改善左室功能。     

Invasive Elektrophysiologie: Komplikationen, Alpträume und deren Management

Most minor side effects of ablation in the right atrium and right ventricle relate to femoral venous catheterization but there is a small risk of severe complications including atrioventricular (AV) block, damage of surrounding structures and thromboembolic events. Impairment of AV conduction can occur during ablation of atrioventricular re-entrant tachycardia, ablation of anteroseptal, mid-septal and parahisian accessory pathways, ablation of ectopic atrial tachycardia originating from the vicinity of the atrioventricular node and when ablating the septal isthmus for typical atrial flutter. Damage of the right coronary artery is a very rare complication after inferior isthmus ablation with high energy. The thromboembolic risk during and after cardioversion and ablation of atrial flutter is higher than previously recognized and anticoagulation therapy decreases this risk. The risk of perforation and tamponade during ablation in the right atrium and right ventricle is very low but particular caution is necessary in thin-walled structures such as the coronary sinus and the upper right ventricular outflow tract. Phrenic nerve injury can be avoided by pacing from the mapping electrode before application of radiofrequency energy at the right atrial free wall. Limitation of power output depending on the site of ablation and titration of energy application with continuous control of temperature and impedance should be considered to minimize the risk of complications.     

经心外膜射频消融治疗心房颤动的实验研究

为探讨射频消融治疗心房颤动（简称房颤）的可行性，对１０条犬进行单心房Ｂｕｒｓｔ刺激和药物＋心房电刺激建立房颤模型并进行心房外膜多条径线的消融。结果显示：消融径线为连续和透壁性损伤；消融后药物＋心房电刺激的房颤诱发率和平均持续时间与消融前比较差异有高度显著性（５８％ｖｓ１００％，４．５ｓｖｓ２０３．５ｓ，Ｐ均＜０．０１）；校正窦房结恢复时间和窦性心率分别较消融前明显延长６５％（１２９±５１ｍｓｖｓ７８±３４ｍｓ）和下降１２％（１５０±６３ｂｐｍｖｓ１７０±３５ｂｐｍ），而房室传导功能和心脏功能无显著性变化。提示射频消融治疗房颤是可行的，其主要机制为消融径线有效地分离阻隔了参与房颤折返环的心房肌群。１条犬消融后发生持续性窦性停搏，可能是损伤窦房结动脉所致     

单纯消融犬心外膜脂肪垫的远期心电生理效应

目的探讨系统消融犬心外膜脂肪垫（完全去迷走神经）的远期心电生理效应。方法成年雄性杂种犬16条,随机分为消融组和假手术组（各8条）。消融组犬经开胸途径消融心外膜所有可视脂肪垫,假手术组仅进行开胸手术。术后8周行心内电生理检查,测定右心房、左心房、左心室、右心室心尖部及右心室流出道等部位的不应期,并进行房性及室性快速心律失常的诱发。结果术后8周实验组基础心率（163．8±12．2）~／min显著高于假手术组对（122．7±13．0）次／min（P〈O．001）;消融组犬的右心房、左心房、左心室、右心室心尖部及右心室流出道不应期分别较假手术组显著缩短（除右心室心尖部P=0．012外,其他部位P〈O．001）,同时该组的快速房性心律失常诱发率显著增加（P=0．04）,两组间的室性快速心律失常诱发率差异无统计学意义。结论仅消融犬心外膜脂肪垫可能具有远期致心律失常作用,尤其在心房水平。     

射频消融改良房室结控制特发性心房颤动所致快速心室率

采用射频消融改良房室结的方法控制７例特发性心房颤动（简称房颤）病人的快速心室率。５例持续性房颤在房颤时消融，２例阵发性房颤在窦性心律时消融，平均放电６±４次，６例成功，１例失败。成功的病例术后复查动态心电图示静息时房颤的平均最大心室率和平均心室率分别从术前的１６５±１１和１３６±１０ｂｐｍ下降到１１１±１４和８８±１１ｂｐｍ（Ｐ均＜０．００１）。平均随访５±４月患者无明显症状，不服药静息心室率均低于１１０ｂｐｍ，有１例阵发性房颤发作显著减少。结果提示：对于症状明显、药物治疗无效的特发性快速房颤的病例，射频消融改良房室结是控制心室率安全和有效的方法。有关机理和远期疗效有待进一步评价     

成熟心耳肌细胞自体移植的实验研究

目的将急性分离的成年兔自体左心耳心肌细胞移植到自体梗死区心肌后,研究其存活情况,以及对心律的影响。方法结扎成年兔的冠状动脉前降支,建立心肌梗死模型,4周后获取自体左心耳组织,急性消化分离为单细胞,经DAPI标记后,分别将细胞悬液和培养基注射到移植组和对照组梗死区内。4周后行心电图检查并取移植区组织进行组织学观察。结果4周后移植组与对照组兔全部存活。心电图检查,移植组心率略高于对照组（P<0.05）,未见异位心律。心肌组织切片见对照组梗死区内为典型心肌梗死后改变,移植组梗死区内有“细胞岛”形成,荧光检测证明移植的心耳肌细胞在移植区存活。结论急性分离的自体左心耳心肌细胞移植到梗死区心肌内可以存活,并不产生异位心律。     

Percutaneous radiofrequency catheter ablation for supraventricular arrhythmias in children

Nineteen procedures were performed in 17 children, aged 10 months to 17 years, using catheter radiofrequency applications for the management of malignant or drug-resistant supraventricular tachyarrhythmias. Diagnoses were junctional ectopic tachycardia in 1 patient, atrioventricular (AV) node reentrant tachycardia in 4 and accessory pathway-mediated tachycardia in 12. Accessory pathway locations were left lateral (n = 4), posteroseptal (n = 3), left posterior (n = 2), right posterolateral (n = 1), right posterior paraseptal (n = 1), right intermediate septal (n = 1) and right anterior (n = 1). Ablation of accessory pathways was performed using 20 to 40 W of energy. The catheter was passed retrograde to the left ventricle in patients with a left-sided pathway and anterograde to the right atrium in those with a right-sided or posteroseptal pathway. In the 12 patients with an accessory pathway, radiofrequency applications were successful in 11 pathways and failed in 2. There were no recurrences of accessory pathway-mediated tachycardia. Atrioventricular node reentrant tachycardia was treated by AV node modification using 15 W of energy applied until first degree AV block occurred. After radiofrequency catheter ablation, there was a prolonged AH interval, tachycardia was not inducible and tachycardia recurred in one patient. For the patient with junctional ectopic tachycardia, 15 to 18 W of energy was delivered at the site of the maximal His bundle electrogram until sinus rhythm and normal AV conduction appeared. After a recurrence, a second procedure abolished tachycardia and AV conduction. In summary, radiofrequency catheter ablation was initially successful in 17 of 19 procedures and ultimately curative in 14 (82%) of 17 patients with no serious complications. Radiofrequency catheter ablation appears to be a safe and effective method for the management of supraventricular tachyarrhythmias in children.     

重组起搏基因质粒构建生物起搏器的实验研究

目的研究旨在构建重组起搏基因质粒pIRES2-EGFP-HCN2,并检测其在体外心房肌细胞及犬病态窦房结综合征(病窦)模型体内的表达。方法对含mHCN2cDNA的PTR载体进行转化和扩增,将所得mHCN2基因定向克隆到真核表达载体pIRES2-EGFP中,进行双酶切来鉴定克隆的正确性。将重组质粒用电穿孔法转染心房肌细胞,同时直接注射至犬病窦模型的窦房结区域,体表心电图监测犬窦房结功能改善情况,并通过荧光显微镜及RT-PCR检测重组质粒pIRES2-EGFP-HCN2在体内外的表达。而对照组仅注射生理盐水。结果构建了重组质粒pIRES2-EGFP-HCN2。荧光显微镜下可见转染后的心房肌细胞呈绿色荧光,其搏动频率较未转染的细胞明显增快[(180±11)次/min与(140±14)次/min,P<0.05]。注射组织的冰冻切片显示绿色荧光蛋白,RT-PCR显示了mHCN2基因片段,体表心电图显示犬窦房结功能改善[(150±13)次/min与(105±17)次/min,P<0.05]。而对照组始终未见到绿色荧光蛋白表达及窦房结功能改善。结论成功构建了重组质粒pIRES2-EGFP-HCN2,并在体外心房肌细胞及犬病窦模型体内成功表达,为生物起搏的研究奠定基础。     

Isolated atrial segment pacing: an alternative to His bundle pacing after atrioventricular junctional ablation.

OBJECTIVES: This study was designed to investigate a practical alternative to His bundle pacing after atrioventricular (AV) junctional ablation by pacing a small area of isolated atrial tissue surrounding the AV node. BACKGROUND: His bundle pacing is preferred after AV junctional ablation in patients with refractory atrial fibrillation. However, it is technically difficult and not clinically useful at the present time. METHODS: This study was conducted in an isolated working swine heart model (n = 5), with real-time imaging capabilities. A small area of atrial tissue surrounding the AV node and the His bundle was isolated using sequential radiofrequency ablation lesions. RESULTS: Complete AV block created by segmental atrial isolation was achieved in 5 of 5 experiments. The isolated atrial segment was bordered by the ablation lines, the tricuspid annulus, and the AV node-His bundle. The AV conduction was characterized using a pacing electrode implanted into the isolated atrial segment. Pacing from the atria, the ventricles, and the isolated atrial segment at different rates confirmed complete bidirectional block between the atria and isolated area, whereas antegrade and retrograde AV nodal conduction between the isolated atrial segment and the ventricles remained intact. Pacing from the isolated area produced minimal changes in systolic left ventricular pressure compared with baseline sinus rhythm (mean -2 mm Hg). CONCLUSIONS: Isolation of a small area of atrial tissue surrounding the AV node is feasible by transcatheter radiofrequency ablation. This procedure may be a useful alternative to conventional AV junctional ablation because it can create complete AV block, while in effect permitting the equivalent of His bundle pacing after AV junctional ablation.     

Ventricular fibrillation occurring after atrioventricular node ablation despite minimal difference between pre- and post-ablation heart rates

We report the case of an 82-year-old man presenting with ventricular fibrillation (VF) occurring acutely after atrioventricular node (AVN) ablation. This patient had severe valvular cardiomyopathy, chronic atrial fibrillation (AF), and underwent prior to the AVN ablation a biventricular implantable cardiac defibrillator positioning. The VF was successfully cardioverted with one external electrical shock. What makes this presentation original is that the pre-ablation spontaneous heart rate in AF was slow (84 bpm), and that VF occurred after ablation despite a minimal heart rate drop of only 14 bpm. VF is the most feared complication of AVN ablation, but it had previously only been described in case of acute heart rate drop after ablation of at least 30 bpm (and more frequently > 50 bpm). This case report highlights the fact that VF may occur after AVN ablation regardless of the heart rate drop, rendering temporary fast ventricular pacing mandatory whatever the pre-ablation heart rate.