兔缓慢性心律失常模型建立与MSCs同种异体心脏移植后细胞整合及功能研究
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摘要
研究背景与目的：病态窦房结综合征（病窦综合征）是一种常见临床疾病，该疾病对人类健康危害很大。目前，关于病窦综合征的发病机理可能有以下几种：1）退行性变；2）心肌病；3）心肌炎症；4）手术损伤等，植入永久性人工电子心脏起搏器仍然是目前临床上主要的治疗手段。电子心脏起搏器有一些固有缺陷，如：电池寿命有限、需要定期检测及更换，价格昂贵，可能发生电极断裂及全身严重感染等并发症。因此，探讨如何建立一个具有正常生理起搏功能的“生物起搏器”替代窦房结或者其他传导系统细胞发挥功能，从而避免永久性人工电子心脏起搏器植入，已经成为目前心脏电生理学界研究的重点及热点内容之一，部分学者在以基因治疗和细胞移植技术为平台构建心脏生物起搏器治疗缓慢性心律失常方面进行了不少有益的探索。部分研究将骨髓间充质干细胞（mesenchymal stem cells, MSCs）经基因修饰后移植或者直接移植、胚胎干细胞（embryonic stem cells）体外诱导分化为起搏细胞进行移植或者成体心肌细胞直接移植至生物体内试图建立新的心脏起搏点，但其结果并不十分理想，其主要问题包括：1）胚胎干细胞的伦理学问题；2）干细胞诱导分化技术不成熟；3）心率偏慢、移植细胞体内存活时间不确定以及较低的心率变异性；4）成体心肌细胞来源比较困难等。
超极化激活的环核苷酸门控的阳离子通道（hyperpolarization activated cyclicnucleotide gated cation channel，HCN）基因是起搏离子流(funny current, If)的分子基础；HCN基因家族四个成员HCN1~HCN4中HCN4基因与If的形成关系最为密切。近年来的研究已经证实起搏电流If在调控心脏和神经元的自发起搏活动中起着非常重要的作用。目前正在进行的以基因治疗和细胞移植技术为平台构建心脏生物起搏器治疗缓慢性心律失常的研究中，HCN通道基因已成为备受重视的备选基因。在已克隆的4种HCN基因亚型中，HCN4主要分布于心脏的特殊传导系统组织中。既往研究均选用大型实验动物犬/猪，采用射频消融法获得永久性三度房室传导阻滞模型，所选靶基因多为HCN2基因，本研究旨在探讨经化学消融窦房结的基础上行双侧迷走神经刺激制备一过性兔缓慢性心律失常模型，观察mHCN4基因修饰的兔骨髓间充质干细胞移植至兔左室游离壁心外膜下之后的在体整合及起搏功能情况，为探讨与完善生物起搏治疗提供新的研究思路与方法。
方法：
1．取2月龄日本大耳白兔无菌条件下行股骨粗隆穿刺抽取骨髓，采用Percoll梯度密度离心法洗涤离心2遍后，收集中间层呈云雾状白膜的单个核细胞层，用DMEM培养基洗涤两次，加入胎牛血清(10%)的DMEM培养基中按照10×108/ml常规行接种、培养及传代。选取生长状态良好的第4代兔MSCs细胞按2×104/孔的接种密度接种于24孔板中，将培养基与含有pMSCV-mHCN4-EGFP的病毒上清按l：1比例稀释后加入铺板的兔MSCs细胞中，并加入polybrene（终浓度调至2μg/ml）。转染24h后，用2μg/m1的嘌呤霉素加压筛选培养10～15天，即可获得mHCN4感染阳性的兔MSCs。免疫荧光法检测空白对照、EGFP组及mHCN4组细胞目的基因表达情况。以膜片钳技术对转染表达的mHCN4通道进行电生理学测定。
2.采用化学消融兔窦房结的基础上双侧迷走神经程序刺激法制备一过性缓慢性心律失常模型。化学消融方案：以干棉签拭干右心耳和上腔静脉交界处区域（即SAN区），用大小约5mm×5mm×5mm棉签蘸取20％的甲醛溶液置于SAN区湿敷。动态监测ECG变化情况并记录HR变化，以湿敷后HR下降超过30％或者出现交界性逸搏、窦性停搏为消融成功标准，停止湿敷，持续监测ECG至少2h，如ECG显示心率/节律出现反复，则再次行甲醛湿敷直至达标为止。迷走刺激方案：采用频率分级递增法进行单/双侧迷走神经刺激。选取刺激电压4~6V，刺激频率依次为：2.5Hz、5Hz、10Hz、15Hz以及20Hz（1Hz=60次/分）。先行2.5Hz刺激，时间不超过60s，经刺激达到刺激终点时对观察指标进行记录；如刺激60s仍没有达到刺激终点，则增加刺激频率进入下一档刺激。两次刺激间隔时间不少于180s。以此类推。达到刺激终点的实验兔在间隔180s后行120s持续刺激。
3.移植后3天、1周、2周及4周分别于麻醉下化学消融窦房结后程序刺激双侧迷走神经，观察有无左室移植细胞来源的早搏或室性节律。术后取材行HE染色观察细胞形态，DAB显色检测缝隙连接蛋白表达，免疫荧光检测EGFP及mHCN4基因表达。
结果：
1．成功将携带mHCN4基因的逆转录病毒载体（pMSCV-mHCN4-EGFP）转染至兔MSCs，经2μg/mL的嘌呤霉素加压筛选后获得了可稳定表达mHCN4基因的兔MSCs。
2．转染mHCN4基因的兔MSCs可以记录到具有明显时间及电压依赖特性的对细胞外Cs+敏感的超极化激活的内向电流，其在-140mV指令电压下的平均电流密度为-42.8±3.6pA/pF；在指令电压的末端去极化至+20mV时可以记录到明显的尾电流；而EGFP对照组未能检测到明显的超极化内向电流。
3．经化学消融窦房结后实验兔基础心率显著下降（307±21次/分vs126±28次/分，p<0.01）。以不同频率刺激右侧迷走神经均表现为心率减慢，刺激左侧迷走神经也以心率减慢为主，随刺激频率增加可出现窦性停搏及交界性逸搏心律。以10Hz以上刺激频率同时刺激双侧迷走神经可以出现窦性停搏及室性逸搏，部分出现3度房室传导阻滞。随着双侧迷走神经刺激频率的增加，达到刺激终点所需的刺激时间逐渐缩短。所有实验兔在终止刺激后均能逐渐恢复刺激前窦性节律，但恢复时间随刺激频率增加而延长。
4.经化学消融窦房结基础上迷走神经刺激抑制窦性节律条件下，移植后3天时各组实验兔出现的室性逸搏心率无统计学差异；移植后1周mHCN4组仅有1只出现高于对照组及EGFP组心率的移植细胞来源的室性节律；移植后2周及4周时，mHCN4组室性心率显著高于对照组及EGFP组，QRS波时间也显著短于对照组及EGFP组。
5.随着时间延长细胞移植区HE染色呈现出明显的动态演变：移植后3天时移植细胞多为圆形并成簇聚集，与周围正常心室肌界限明显；移植后1周时移植细胞数量明显减少，与正常心室肌相邻处可见少量细胞呈短梭形；移植后2周时移植细胞与周围心室肌过渡较为自然，梭形细胞数量增多；移植后4周时存活移植细胞形态基本呈长梭形，与周围心室肌界限较模糊。对照组注射等体积培养基后移植部位组织切片HE染色表现为轻微炎症反应，动态演变不明显。DAB显色可见移植细胞与宿主细胞之间Cx43、Cx45均有表达，呈褐色，线、颗粒状。
6.移植细胞在移植术后3d时即可检出EGFP和mHCN4阳性表达，但细胞基本呈圆形，尚未伸展开，Cx43、Cx45表达为阴性。移植后1周时存活细胞减少，少量细胞呈短梭形，EGFP、mHCN4及Cx43、Cx45表达均为阳性。移植后2周时移植细胞伸展为长梭形，形态接近于相邻心室肌细胞，可见移植细胞与宿主细胞之间Cx43、Cx45阳性表达。移植后4周时镜下表现与移植后2周相似。
结论：
1．mHCN4基因可成功转染至兔MSCs，经嘌呤霉素加压筛选后可稳定表达。
2．mHCN4基因转染修饰的兔MSCs可成功记录到If电流，具备心脏起搏细胞的电生理特性。
3．采用化学消融窦房结基础上双侧迷走神经刺激法可以成功制备缓慢性心律失常模型，能够满足生物起搏在体实验研究要求。
4．mHCN4基因修饰的兔MSCs左室游离壁心外膜下移植后移植细胞形态呈明显动态演变，移植细胞与宿主细胞之间Cx43、Cx45均有表达，呈线、颗粒状；可与心肌细胞构成功能性的缝隙连接，进行有效的胞间通讯。
5. mHCN4基因修饰的兔MSCs同种异体移植后约1周左右可表现出起搏功能，移植后2周及4周时，mHCN4组室性心率显著高于对照组及EGFP组，QRS波时间也显著短于对照组及EGFP组。移植细胞起搏功能于2周时观察可稳定发挥作用，至移植后4周仍持续存在。
Backgrounds and objections:
Sick sinus syndrome(SSS) is a frequent clinical syndrome, which have a great healthhazards to human. The pathological mechanisms of SSS such as degeneration,cardiomyopathy, inflammation of myocardium, and damage of operation is not very cleartill now and there are not very effective prevent and treatment methods with it. Althoughimplantation of permanent artificial electric cardiac pacemaker is still the current maintreatment, that is not good enough. Among their shortcomings are limited battery life, theneed for examination regularly, high cost, possible complications of permanent catheterimplantation into the heart such as electrode breakage, severe infection. So cardiacelectrophysiologists or knowledgeable physicians focus on how to build a biologicalpacemaker with the normal physiological function as the substitute for the electronic heartpacemaker. Some researchers has used the gene therapy and cell transplantation to build aexperimental biological pacemaking platform for bradyarrhythmia Some approaches havebeen attempted to establish biological pacemaker by transplanting gene modifiedmesenchymal stem cells (MSCs) or MSCs alone, embryonic stem cells induced in vitro,andcardiac myocyte into animal hearts. These approaches are not optimal enough because ofethical problems, immature technology of stem csll inducing differentiation, the low pacingrate, the uncertain and short duration of pacing function, the low heart rate variability, poorsource of cardiac myocyte, and so on.
Hyperpolarization activated cyclic nucleotide gated cation channel (HCN) is themolecular basis of funny current (If). HCN4as1/4members in the family of Ifchannelsmaybe a optimal biological pacemaking target gene because it is essential for modulation ofIf. Recently, Ifwas shown to play an important role in the spontaneous pacing activity ofcardiomyocytes and neurons. HCN genes are becoming important candidate genes in investigations using biological pacemakers to treat bradyarrhythmia, which were based ongene therapy and cell transplantation techniques. In cloned4HCN gene subtypes, HCN4ismainly expressed in the cardiac specific conduction system. Previously,3atrioventricularblock models by radiofrequency catheter were established on a large animal model likedogs or pigs, and HCN2was the target gene. In this study, we established a rabbitbradyarrhythmia model based on chemical ablation of the sinoatrial node and sequentialbilateral vagus nerve stimulation, and evaluated the integration and pacing function aftermHCN4-modified rabbit BMSC were transplanted into left ventricle epicardium. This studyprovides new insights and methods to explore biological pacing therapy.
Methods
1. BMSCs were separated and purified with Percoll separating medium by densitygradient centrifugation and adherence method. Bone marrow was obtained from2month-old Japanese white rabbits via intertrochanteric aspiration under aseptic conditionsand washed twice by Percoll density gradient centrifugation. The monocytes in middlewhite cloudy layer were harvested and washed twice with DMEM at1,000rpm for5minutes. Cells were cultured and propagated in DMEM containing10%fetal bovine serumat a concentration of10×108/ml. Rabbit MSCs (p=4) were plated into a24-well plate at2×104/well, a supernatant containing pMSCV-mHCN4-EGFP virus was mixed withmedium at1:1and added into rabbit MSCs together with polybrene at a final concentrationof2μg/ml. Twenty-four hours after transduction puromycin at2μg/ml was added and cellswere selected for10~15days to get transduced rabbit MSCs. Immunofluorescence wasused to determine the gene expression level in the control group, EGFP group, and mHCN4group.The electronic characters of If channels were detected by whole cell patch clamp.
2. Establishing a bradyarrhythmia rabbit model by sinus node chemical ablation andbilateral vagus nerve stimulation. Methods of chemical ablation: after drying the junction ofsuperior vena cava and right atrial appendage (SAN area), placed the5mm×5mm×5mmcotton with20%formaldehyde at SAN. Stopped the chemical stimulation if HR decreasedby30%, or sinus arrest occurred, or junctional escape occurred. ECGs are still record for2h. If ECG appeared repeatedly, then use the chemical stimulation at SAN again until HRdecreases by30%, or sinus arrest occurres,or junctional escape occurres. Methods of vagusnerve stimulation: incremental classification method was used to perform the vagus nerve stimulation with electrical rectangular pulses in4~6V stimulating voltage at2.5Hz,5Hz,10Hz,15Hz,20Hz stimulus frequency (1Hz=60beats/min), respectively. Thestimulation was performed with pulses at10Hz at first. And the relative indexes, includingHR, time for reaching the the stimulus endpoint, recovery time and so on, were recorded ifthe rabbit arrived at the stimulus endpoint in60s. If rabbit doesn’t arrive in60s, then thenext stimulation with180s intervals was performed until it reached the stimulus endpoint.180s later after reaching the stimulus endpoint, the rabbit received the stimulation for120sagain.
3. At3days,1week,2weeks and4weeks after transplantation, chemical ablation ofthe sinoatrial node was performed and bilateral vagus nerves were sequentially stimulatedto observe premature left ventricular contraction or left ventricular rhythm. Cellmorphology and gap junction were measured by HE and DAB staining, respectively. EGFPand mHCN4expression levels were determined by immunofluorescence..
Results
1. Rabbit MSCs were successfully transfected with pMSCV-mHCN4-EGFP, whichcan stably expressing mHCN4gene after selected by puromysin with a concentration of2μg/mL.
2. Hyperpolarization activated inward current which was time and voltage dependentand sensitive to extrocelluar Cs+could be detected in rabbit MSCs transduced withmHCN4. The average current density was-42.8±3.6pA/pF under a reference voltage of-140mV. Apparent tail current could be detected when reference voltage was+20mV.Meanwhile, this hyperpolarization activated inward current could not be detected in EGFPcontrol group under the same conditions.
3. Basic heart rate significantly dropped down in those rabbits after chemical ablationwith20%formaldehyde (307±21beats/min vs126±28beats/min, p <0.01). Heart rate ofeach rabbit slowed down with the right vagus nerve stimulation at different pulsefrequencies. Sinus arrest and junctional escape besides heart rate slowed down occurredwith the increasing-frequency pulse stimulation with the left vagus nerve. Sinus arrest andventricular escape beat occurred with the bilateral vagus nerve stimulation at10Hz or more.Some cases were that3-degree atrioventricular block. It spent less time in reaching the thestimulus endpoint with the increase of pulse frequency for bilateral vagus nerve stimulation. The sinus rhythm of all rabbits restored to normal as before stimulation. But recovery timewas needed with the increase of pulse frequency.
4. No significant difference in ventricular escape rhythm was observed after chemicalablation of the sinoatrial node and sequential bilateral vagus nerve stimulation among thethree groups at3days after transplantation. At1week after transplantation, only one rabbitin the mHCN4group had a ventricular rhythm higher than the control group or EGFP group.However, the mHCN4group had significantly higher ventricular rhythm and shorter QRSduration than the control or EGFP group, respectively.
5. Morphology of the transplanted cells dynamically changed as observed by HEstaining over time: At3days after transplantation, transplanted cells were round andclustered and had well-defined boundaries separating them from the adjacent normalventricle myocytes. At1week after transplantation, the cells decreased significantly, andshort spindle cells could be observed in the region adjacent to the normal ventriclemyocytes. At2weeks after transplantation, the transplanted cells displayed gradualtransition to the adjacent ventricle myocytes and spindle cells increased. At4weeks aftertransplantation, the surviving transplanted cells were in long-spindle morphology and couldnot be clearly discriminated from adjacent ventricle myocytes. In the control group, mildinflammation was observed in the transplanted region as determined by HE staining aftermedium injection, and no dynamic changes were observed. Cx43and Cx45could bedetected between transplanted cells and host cells by DAB staining, which were brownlinear granulates.
6. EGFP and mHCN4could be detected in the transplanted cells at3days aftertransplantation. The cells were round, not spread, and were negative for Cx43and Cx45.One week after transplantation, surviving transplanted cells decreased, a few were short andspindle-like, and positive for EGFP, mHCN4, Cx43and Cx45. Two weeks aftertransplantation, transplanted cells were long and spindle-like, with morphology similar tothe adjacent ventricle myocytes. Cx43and Cx45could be detected between transplantedcells and host cells. Similar results could be observed4weeks after transplantation.
Conclusions
1. mHCN4gene can be successfully transfected into rabbit MSCs, and can stablyexpress after selected by puromysin.
2. Ifcan be detected from rabbit MSCs transfeced with mHCN4gene which have theelectrophysiological ability of cardiac pacemaker cells.
3. A bradyarrhythmia model can be successfully established by chemical ablation ofthe sinoatrial node and sequential bilateral vagus nerve stimulation. This model can be usedto study biological pacing in vivo.
4. The mHCN4-modified rabbit MSCs displayed evident dynamic morphologychanges after being transplanted into rabbit left ventricle free wall epicardium. Cx43andCx45could be detected between transplanted cells and host cells, which were brown lineargranulates.
5. The MSCs showed pacing function approximately1week after transplantation.The mHCN4-transduced MSC group had a significantly higher ventricular rate and a shorter QRS duration than that of the control and EGFP group at2and4weeks aftertransplantation. The pacing cells worked well at2weeks and persisted stably4weeks aftertransplantation.
超极化激活的环核苷酸门控的阳离子通道（hyperpolarization activated cyclicnucleotide gated cation channel，HCN）基因是起搏离子流(funny current, If)的分子基础；HCN基因家族四个成员HCN1~HCN4中HCN4基因与If的形成关系最为密切。近年来的研究已经证实起搏电流If在调控心脏和神经元的自发起搏活动中起着非常重要的作用。目前正在进行的以基因治疗和细胞移植技术为平台构建心脏生物起搏器治疗缓慢性心律失常的研究中，HCN通道基因已成为备受重视的备选基因。在已克隆的4种HCN基因亚型中，HCN4主要分布于心脏的特殊传导系统组织中。既往研究均选用大型实验动物犬/猪，采用射频消融法获得永久性三度房室传导阻滞模型，所选靶基因多为HCN2基因，本研究旨在探讨经化学消融窦房结的基础上行双侧迷走神经刺激制备一过性兔缓慢性心律失常模型，观察mHCN4基因修饰的兔骨髓间充质干细胞移植至兔左室游离壁心外膜下之后的在体整合及起搏功能情况，为探讨与完善生物起搏治疗提供新的研究思路与方法。
方法：
1．取2月龄日本大耳白兔无菌条件下行股骨粗隆穿刺抽取骨髓，采用Percoll梯度密度离心法洗涤离心2遍后，收集中间层呈云雾状白膜的单个核细胞层，用DMEM培养基洗涤两次，加入胎牛血清(10%)的DMEM培养基中按照10×108/ml常规行接种、培养及传代。选取生长状态良好的第4代兔MSCs细胞按2×104/孔的接种密度接种于24孔板中，将培养基与含有pMSCV-mHCN4-EGFP的病毒上清按l：1比例稀释后加入铺板的兔MSCs细胞中，并加入polybrene（终浓度调至2μg/ml）。转染24h后，用2μg/m1的嘌呤霉素加压筛选培养10～15天，即可获得mHCN4感染阳性的兔MSCs。免疫荧光法检测空白对照、EGFP组及mHCN4组细胞目的基因表达情况。以膜片钳技术对转染表达的mHCN4通道进行电生理学测定。
2.采用化学消融兔窦房结的基础上双侧迷走神经程序刺激法制备一过性缓慢性心律失常模型。化学消融方案：以干棉签拭干右心耳和上腔静脉交界处区域（即SAN区），用大小约5mm×5mm×5mm棉签蘸取20％的甲醛溶液置于SAN区湿敷。动态监测ECG变化情况并记录HR变化，以湿敷后HR下降超过30％或者出现交界性逸搏、窦性停搏为消融成功标准，停止湿敷，持续监测ECG至少2h，如ECG显示心率/节律出现反复，则再次行甲醛湿敷直至达标为止。迷走刺激方案：采用频率分级递增法进行单/双侧迷走神经刺激。选取刺激电压4~6V，刺激频率依次为：2.5Hz、5Hz、10Hz、15Hz以及20Hz（1Hz=60次/分）。先行2.5Hz刺激，时间不超过60s，经刺激达到刺激终点时对观察指标进行记录；如刺激60s仍没有达到刺激终点，则增加刺激频率进入下一档刺激。两次刺激间隔时间不少于180s。以此类推。达到刺激终点的实验兔在间隔180s后行120s持续刺激。
3.移植后3天、1周、2周及4周分别于麻醉下化学消融窦房结后程序刺激双侧迷走神经，观察有无左室移植细胞来源的早搏或室性节律。术后取材行HE染色观察细胞形态，DAB显色检测缝隙连接蛋白表达，免疫荧光检测EGFP及mHCN4基因表达。
结果：
1．成功将携带mHCN4基因的逆转录病毒载体（pMSCV-mHCN4-EGFP）转染至兔MSCs，经2μg/mL的嘌呤霉素加压筛选后获得了可稳定表达mHCN4基因的兔MSCs。
2．转染mHCN4基因的兔MSCs可以记录到具有明显时间及电压依赖特性的对细胞外Cs+敏感的超极化激活的内向电流，其在-140mV指令电压下的平均电流密度为-42.8±3.6pA/pF；在指令电压的末端去极化至+20mV时可以记录到明显的尾电流；而EGFP对照组未能检测到明显的超极化内向电流。
3．经化学消融窦房结后实验兔基础心率显著下降（307±21次/分vs126±28次/分，p<0.01）。以不同频率刺激右侧迷走神经均表现为心率减慢，刺激左侧迷走神经也以心率减慢为主，随刺激频率增加可出现窦性停搏及交界性逸搏心律。以10Hz以上刺激频率同时刺激双侧迷走神经可以出现窦性停搏及室性逸搏，部分出现3度房室传导阻滞。随着双侧迷走神经刺激频率的增加，达到刺激终点所需的刺激时间逐渐缩短。所有实验兔在终止刺激后均能逐渐恢复刺激前窦性节律，但恢复时间随刺激频率增加而延长。
4.经化学消融窦房结基础上迷走神经刺激抑制窦性节律条件下，移植后3天时各组实验兔出现的室性逸搏心率无统计学差异；移植后1周mHCN4组仅有1只出现高于对照组及EGFP组心率的移植细胞来源的室性节律；移植后2周及4周时，mHCN4组室性心率显著高于对照组及EGFP组，QRS波时间也显著短于对照组及EGFP组。
5.随着时间延长细胞移植区HE染色呈现出明显的动态演变：移植后3天时移植细胞多为圆形并成簇聚集，与周围正常心室肌界限明显；移植后1周时移植细胞数量明显减少，与正常心室肌相邻处可见少量细胞呈短梭形；移植后2周时移植细胞与周围心室肌过渡较为自然，梭形细胞数量增多；移植后4周时存活移植细胞形态基本呈长梭形，与周围心室肌界限较模糊。对照组注射等体积培养基后移植部位组织切片HE染色表现为轻微炎症反应，动态演变不明显。DAB显色可见移植细胞与宿主细胞之间Cx43、Cx45均有表达，呈褐色，线、颗粒状。
6.移植细胞在移植术后3d时即可检出EGFP和mHCN4阳性表达，但细胞基本呈圆形，尚未伸展开，Cx43、Cx45表达为阴性。移植后1周时存活细胞减少，少量细胞呈短梭形，EGFP、mHCN4及Cx43、Cx45表达均为阳性。移植后2周时移植细胞伸展为长梭形，形态接近于相邻心室肌细胞，可见移植细胞与宿主细胞之间Cx43、Cx45阳性表达。移植后4周时镜下表现与移植后2周相似。
结论：
1．mHCN4基因可成功转染至兔MSCs，经嘌呤霉素加压筛选后可稳定表达。
2．mHCN4基因转染修饰的兔MSCs可成功记录到If电流，具备心脏起搏细胞的电生理特性。
3．采用化学消融窦房结基础上双侧迷走神经刺激法可以成功制备缓慢性心律失常模型，能够满足生物起搏在体实验研究要求。
4．mHCN4基因修饰的兔MSCs左室游离壁心外膜下移植后移植细胞形态呈明显动态演变，移植细胞与宿主细胞之间Cx43、Cx45均有表达，呈线、颗粒状；可与心肌细胞构成功能性的缝隙连接，进行有效的胞间通讯。
5. mHCN4基因修饰的兔MSCs同种异体移植后约1周左右可表现出起搏功能，移植后2周及4周时，mHCN4组室性心率显著高于对照组及EGFP组，QRS波时间也显著短于对照组及EGFP组。移植细胞起搏功能于2周时观察可稳定发挥作用，至移植后4周仍持续存在。
Backgrounds and objections:
Sick sinus syndrome(SSS) is a frequent clinical syndrome, which have a great healthhazards to human. The pathological mechanisms of SSS such as degeneration,cardiomyopathy, inflammation of myocardium, and damage of operation is not very cleartill now and there are not very effective prevent and treatment methods with it. Althoughimplantation of permanent artificial electric cardiac pacemaker is still the current maintreatment, that is not good enough. Among their shortcomings are limited battery life, theneed for examination regularly, high cost, possible complications of permanent catheterimplantation into the heart such as electrode breakage, severe infection. So cardiacelectrophysiologists or knowledgeable physicians focus on how to build a biologicalpacemaker with the normal physiological function as the substitute for the electronic heartpacemaker. Some researchers has used the gene therapy and cell transplantation to build aexperimental biological pacemaking platform for bradyarrhythmia Some approaches havebeen attempted to establish biological pacemaker by transplanting gene modifiedmesenchymal stem cells (MSCs) or MSCs alone, embryonic stem cells induced in vitro,andcardiac myocyte into animal hearts. These approaches are not optimal enough because ofethical problems, immature technology of stem csll inducing differentiation, the low pacingrate, the uncertain and short duration of pacing function, the low heart rate variability, poorsource of cardiac myocyte, and so on.
Hyperpolarization activated cyclic nucleotide gated cation channel (HCN) is themolecular basis of funny current (If). HCN4as1/4members in the family of Ifchannelsmaybe a optimal biological pacemaking target gene because it is essential for modulation ofIf. Recently, Ifwas shown to play an important role in the spontaneous pacing activity ofcardiomyocytes and neurons. HCN genes are becoming important candidate genes in investigations using biological pacemakers to treat bradyarrhythmia, which were based ongene therapy and cell transplantation techniques. In cloned4HCN gene subtypes, HCN4ismainly expressed in the cardiac specific conduction system. Previously,3atrioventricularblock models by radiofrequency catheter were established on a large animal model likedogs or pigs, and HCN2was the target gene. In this study, we established a rabbitbradyarrhythmia model based on chemical ablation of the sinoatrial node and sequentialbilateral vagus nerve stimulation, and evaluated the integration and pacing function aftermHCN4-modified rabbit BMSC were transplanted into left ventricle epicardium. This studyprovides new insights and methods to explore biological pacing therapy.
Methods
1. BMSCs were separated and purified with Percoll separating medium by densitygradient centrifugation and adherence method. Bone marrow was obtained from2month-old Japanese white rabbits via intertrochanteric aspiration under aseptic conditionsand washed twice by Percoll density gradient centrifugation. The monocytes in middlewhite cloudy layer were harvested and washed twice with DMEM at1,000rpm for5minutes. Cells were cultured and propagated in DMEM containing10%fetal bovine serumat a concentration of10×108/ml. Rabbit MSCs (p=4) were plated into a24-well plate at2×104/well, a supernatant containing pMSCV-mHCN4-EGFP virus was mixed withmedium at1:1and added into rabbit MSCs together with polybrene at a final concentrationof2μg/ml. Twenty-four hours after transduction puromycin at2μg/ml was added and cellswere selected for10~15days to get transduced rabbit MSCs. Immunofluorescence wasused to determine the gene expression level in the control group, EGFP group, and mHCN4group.The electronic characters of If channels were detected by whole cell patch clamp.
2. Establishing a bradyarrhythmia rabbit model by sinus node chemical ablation andbilateral vagus nerve stimulation. Methods of chemical ablation: after drying the junction ofsuperior vena cava and right atrial appendage (SAN area), placed the5mm×5mm×5mmcotton with20%formaldehyde at SAN. Stopped the chemical stimulation if HR decreasedby30%, or sinus arrest occurred, or junctional escape occurred. ECGs are still record for2h. If ECG appeared repeatedly, then use the chemical stimulation at SAN again until HRdecreases by30%, or sinus arrest occurres,or junctional escape occurres. Methods of vagusnerve stimulation: incremental classification method was used to perform the vagus nerve stimulation with electrical rectangular pulses in4~6V stimulating voltage at2.5Hz,5Hz,10Hz,15Hz,20Hz stimulus frequency (1Hz=60beats/min), respectively. Thestimulation was performed with pulses at10Hz at first. And the relative indexes, includingHR, time for reaching the the stimulus endpoint, recovery time and so on, were recorded ifthe rabbit arrived at the stimulus endpoint in60s. If rabbit doesn’t arrive in60s, then thenext stimulation with180s intervals was performed until it reached the stimulus endpoint.180s later after reaching the stimulus endpoint, the rabbit received the stimulation for120sagain.
3. At3days,1week,2weeks and4weeks after transplantation, chemical ablation ofthe sinoatrial node was performed and bilateral vagus nerves were sequentially stimulatedto observe premature left ventricular contraction or left ventricular rhythm. Cellmorphology and gap junction were measured by HE and DAB staining, respectively. EGFPand mHCN4expression levels were determined by immunofluorescence..
Results
1. Rabbit MSCs were successfully transfected with pMSCV-mHCN4-EGFP, whichcan stably expressing mHCN4gene after selected by puromysin with a concentration of2μg/mL.
2. Hyperpolarization activated inward current which was time and voltage dependentand sensitive to extrocelluar Cs+could be detected in rabbit MSCs transduced withmHCN4. The average current density was-42.8±3.6pA/pF under a reference voltage of-140mV. Apparent tail current could be detected when reference voltage was+20mV.Meanwhile, this hyperpolarization activated inward current could not be detected in EGFPcontrol group under the same conditions.
3. Basic heart rate significantly dropped down in those rabbits after chemical ablationwith20%formaldehyde (307±21beats/min vs126±28beats/min, p <0.01). Heart rate ofeach rabbit slowed down with the right vagus nerve stimulation at different pulsefrequencies. Sinus arrest and junctional escape besides heart rate slowed down occurredwith the increasing-frequency pulse stimulation with the left vagus nerve. Sinus arrest andventricular escape beat occurred with the bilateral vagus nerve stimulation at10Hz or more.Some cases were that3-degree atrioventricular block. It spent less time in reaching the thestimulus endpoint with the increase of pulse frequency for bilateral vagus nerve stimulation. The sinus rhythm of all rabbits restored to normal as before stimulation. But recovery timewas needed with the increase of pulse frequency.
4. No significant difference in ventricular escape rhythm was observed after chemicalablation of the sinoatrial node and sequential bilateral vagus nerve stimulation among thethree groups at3days after transplantation. At1week after transplantation, only one rabbitin the mHCN4group had a ventricular rhythm higher than the control group or EGFP group.However, the mHCN4group had significantly higher ventricular rhythm and shorter QRSduration than the control or EGFP group, respectively.
5. Morphology of the transplanted cells dynamically changed as observed by HEstaining over time: At3days after transplantation, transplanted cells were round andclustered and had well-defined boundaries separating them from the adjacent normalventricle myocytes. At1week after transplantation, the cells decreased significantly, andshort spindle cells could be observed in the region adjacent to the normal ventriclemyocytes. At2weeks after transplantation, the transplanted cells displayed gradualtransition to the adjacent ventricle myocytes and spindle cells increased. At4weeks aftertransplantation, the surviving transplanted cells were in long-spindle morphology and couldnot be clearly discriminated from adjacent ventricle myocytes. In the control group, mildinflammation was observed in the transplanted region as determined by HE staining aftermedium injection, and no dynamic changes were observed. Cx43and Cx45could bedetected between transplanted cells and host cells by DAB staining, which were brownlinear granulates.
6. EGFP and mHCN4could be detected in the transplanted cells at3days aftertransplantation. The cells were round, not spread, and were negative for Cx43and Cx45.One week after transplantation, surviving transplanted cells decreased, a few were short andspindle-like, and positive for EGFP, mHCN4, Cx43and Cx45. Two weeks aftertransplantation, transplanted cells were long and spindle-like, with morphology similar tothe adjacent ventricle myocytes. Cx43and Cx45could be detected between transplantedcells and host cells. Similar results could be observed4weeks after transplantation.
Conclusions
1. mHCN4gene can be successfully transfected into rabbit MSCs, and can stablyexpress after selected by puromysin.
2. Ifcan be detected from rabbit MSCs transfeced with mHCN4gene which have theelectrophysiological ability of cardiac pacemaker cells.
3. A bradyarrhythmia model can be successfully established by chemical ablation ofthe sinoatrial node and sequential bilateral vagus nerve stimulation. This model can be usedto study biological pacing in vivo.
4. The mHCN4-modified rabbit MSCs displayed evident dynamic morphologychanges after being transplanted into rabbit left ventricle free wall epicardium. Cx43andCx45could be detected between transplanted cells and host cells, which were brown lineargranulates.
5. The MSCs showed pacing function approximately1week after transplantation.The mHCN4-transduced MSC group had a significantly higher ventricular rate and a shorter QRS duration than that of the control and EGFP group at2and4weeks aftertransplantation. The pacing cells worked well at2weeks and persisted stably4weeks aftertransplantation.
引文
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