应用经皮心内膜注射导管在体移植生物起搏器的实验研究
|
摘要
目的
缓慢型心律失常是临床上常见的心律失常类型,严重影响患者的生活质量和生命安全。20世纪50年代,心脏植入性电子起搏装置的发明,为该病的治疗带来了革命性的变化。然而,心脏电子起搏器终究只是一种缓解症状的治疗方法,在长期的使用过程中也出现了诸多缺陷。心脏起搏生理的基础研究方面的进展,为起搏功能障碍的治疗开辟了新的前景,生物起搏器的概念也应运而生。近十余年以来,生物起搏器的研究取得了巨大进展。目前,生物起搏器的体内实验主要通过外科开胸手术的途径进行移植,存在创伤大、定位效果差等缺点,因此移植途径是制约生物起搏器临床应用的重要因素之一。探索适宜的移植手段是促进生物起搏进入临床实验阶段所需要克服的重要问题。本课题以犬的骨髓间充质干细胞(Bone-Marrow Mesenchymal Stem Cells, BM-MSCs)作为基因转染的靶细胞,构建超极化激活环核苷酸门控阳离子通道(Hyperpolarization-activated Cyclic Nucleotide-gated channels2, HCN2)基因的重组慢病毒载体,转染BM-MSCs,研究和验证转染后BM-MSCs在体外的起搏功能。采用白行研究设计的经皮心肌内注射导管,通过介入方法将体外构建的生物起搏器移植到实验动物犬的体内,探索该方法的安全性和可行性。
方法
采用密度梯度离心法分离犬的BM-MSCs并进行培养。扩增目的基因mHCN2,构建重组慢病毒表达载体。重组质粒和慢病毒包装系统共同转染T293细胞进行慢病毒的包装。Real-time PCR法检测病毒滴度。Lenti-GFP-HCN2以不同感染复数(multiplication of infection, MOI)转染第3-4代BM-MSCs,确定最佳MOI。设置Lenti-GFP-HCN2转染的BM-MSCs为实验组,Lenti-GFP转染的BM-MSCs为阴性对照组,未转染的BM-MSCs为空白对照组。全细胞膜片钳检测各组BM-MSCs的起搏电流。分离培养新生乳鼠心肌细胞(neonatal rat's cardiomyocytes, NRCs),将各组BM-MSCs和NRCs进行共培养,观察细胞搏动情况,并记录收缩频率。
经颈动脉和股静脉途径分别进入犬的左、右心室,进行龙胆紫稀释液的多点注射,检验自行研究设计的经皮心肌内注射导管的操作性能。将16只健康成年杂种犬(20-25kg)随机分为实验组(注射0.6m1转染了Lenti-GFP-HCN2的MSCs,n=8),阴性对照组(注射0.6m1转染了Lenti-GFP的MSCs, n=4)和空白对照组(注射0.6ml生理盐水,n=4)。经颈动脉途径放入自行设计的注射导管,在左心室室间隔位置定点注射。术后埋植体外遥测的心电记录仪实时监测犬的心率和心律。移植术后2周,对实验动物犬进行双侧迷走神经刺激(S1S1周长100ms/60ms,电压3-5V,时程1min,间隔10min)。记录逸搏心率的频率,分析逸搏来源以及迷走神经刺激后出现逸搏心率的时间间隔。术后2周达实验终点,留取心脏组织标本,冰冻切片后一部分直接在荧光显微镜下观察荧光,一部分进行HE染色以及Cx43,GFP和CD68的免疫荧光染色。
结果
基因测序表明目的基因HCN2已经成功重组至慢病毒表达载体,重组质粒转染T293细胞后,western blot证实在蛋白水平上有HCN2的过表达。本实验中确定慢病毒感染犬BM-MSCs的最佳MOI为20,在转染后48h开始表达绿色荧光,5天达高峰。实验组BM-MSCs可记录到超极化激活的内向电流,而阴性对照组和空白组BM-MSCs在相同记录条件下未能检测到明显的超极化内向电流。实验组BM-MSCs有HCN2蛋白过表达,而阴性对照组和空白组BM-MSCs未见表达。BM-MSCs和NRCs共培养后第3天,实验组NRCs的细胞收缩频率较阴性对照和空白对照组显著提高(P<0.05)。
经皮心肌内注射导管注射的有效性为91.6%,未出现严重并发症。移植术后各组的体外遥测心电记录仪显示室性早搏和室性心动过速的发生在各组间未见显著差异。双侧迷走神经刺激结果发现实验组的异搏心率主要来自注射腔左心室,频率为50.8±3.2bpm,而阴性对照组和空白组的异搏心率主要来自右心室,频率为42.2±6.1bpm(P<0.05)。免疫荧光检测显示介入方法移植到左心室的BM-MSCs在术后2周仍有存活,细胞之间有Cx43的表达和连接,HE染色和CD68的免疫荧光染色显示移植部位并没有显著炎症形成。
结论
mHCN2重组慢病毒(Lenti-GFP-HCN2)转染犬的BM-MSCs能在体外发挥起搏功能。自行研究设计的经皮心肌内注射导管可以用于在体移植转染的BM-MSCs,构建生物起搏器,该方法具有良好的有效性和安全性。利用该导管移植Lenti-GFP-HCN2转染的犬BM-MSCs可以在体内存活,并在移植部位重建起搏点,形成异位搏动。
Objective:
Bradyarrhythmia is a common arrhythmia type which severely affected the patient's health. The invitation of cardiac implanted electronic devices in1950s is a marvel, and advances in this field have been impressive. However, it's a relive therapy rather than cure, and important shortcomings exist. With the further development in cardiac pacing mechanism, the concept of biological pacemaker evolved, and the active investigation in the field of biological pacing is rapidly progressed. However, the in vivo animal studies of biological pacemaker are mainly performed through open thoracotomy, which cause large injury and had poor lacation controlling. Therefore, exploring optimal implantation approaches is very important for further clinic trails. This study used bone-marrow mesenchymal stem cells (BM-MSCs) as transfection target cells, and construct lentiviral expressing vectors carrying mouse Hyperpolarization-activated Cyclic Nucleotide-gated channels2(HCN2) gene. The in vitro pacing function of the transfected BM-MSCs was investigated. A self-designed percutaneous inject catheter was used to deliver the biological pacemaker into the left ventricular of canine, its feasibility and safety was explored. Meanwhile, the in vivo efficiency of the biological pacemaker was tested.
Methods:
Canine BM-MSCs was isolated by density gradient centrifugation and cultured. mHCN2gene fragment was amplified through PCR, then was connected with lentiviral vector. T293cells were con-transfected with lentiviral vector and packaging systems. The titers of lentiviral vectors were tested by real-time PCR. Gradient MOI of5,10,15,20,25,30was performed to determine the optimal MOI for the transfection of canine BM-MSCs. Set experiment group (Lenti-GFP-HCN2transfection), negative control group (Lenti-GFP transfection) and blank control group (with lentivirus transfection). Whole cell patch clamp tested the pacing current and western blot tested the HCN2protein expression of BM-MSCs in each group. NRCs were isolated and cultured, BM-MSCs was co-cultured with NRCs, the contraction frequency was observed and recorded. Catheter was inserted via carotid artery and femoral vein into left and right ventricle separately, diluted gentian violet was muitidot injected into the ventricle to testify the manipulation properties of the inject catheter.16health adult mongrel canine (20-25kg) were randomly divided into experiment group (0.6ml Lenti-GFP-HCN2 transfected MSCs was administrated, n=8), negative control group (0.6ml Lenti-GFP transfected MSCs was administrated, n=4), and blank control group (0.6ml saline was administrated, n=4). Inject catheter was inserted via carotid artery into left ventricle, septum inject was performed under the instruction of X-ray. Telemetering ECG recorder was implanted underneath the skin for heart rhythm monitoring.2weeks later, bilateral vague nerve stimulation was conducted (S1S1100ms/60ms, voltage3-5V, duration1min, interval10min). The escaping rhythm was recorded, its origin and frequency was analyzed. Heart tissue was obtained when end point was reached, frozen sections were sliced for GFP fluorescence, immunoflourescens stain and HE stain.
Results:
DNA sequencing demonstrated that mHCN2recombinated lentiviral vectors were constructed successfully. Western blot test confirmed that there was mHCN2protein expression in the T293cells, which was transfected with the mHCN2recombinated lentiviral vectors. The optimal lentivirus transfection MOI for canine BM-MSCs was20. Patch clamp showed the production of hyperpolarization-activated inward currents in experiment group while not in negative or blank control group. There was a high HCN2protein expression in experiment group other than negative and blank control groups.3days after co-culture, NRCs in experiment group revealed a significantly higher contraction frequency (P<0.05). The injection efficiency was calculated as91.6%, there was no severe complication associated with endomyocardial injection operation. Telemetering ECG recorder revealed premature ventricular contraction and ventricular tachycardia, and no significant differences were observed between three groups. Bilateral vague nerve stimulation showed that in experiment group, the escape rhythm mainly originated from left ventricle with an average frequency of50.8±3.2bpm, while the escape rhythm of negative and blank control groups from right ventricle, average frequency was42.2±6.1bpm (P<0.05). Histopathological examination proved that the injected BM-MSCs survived in vivo, there were Cx43connected between cells, HE stain and CD68immunoflourescens stain indicated no marked inflammation.
Conclusions:
In vitro transfection of canine BM-MSCs with Lenti-GFP-HCN2produced pacing current and expressed HCN2protein, indicating a potential of pacing function. The self-designed percutaneous endomyocardial inject catheter was proved to have highly-qualified efficiency and safety, it provided a better delivery for biological pacemaker study. Lenti-GFP-HCN2transfected canine BM-MSCs can be successfully delivered by this catheter, cells survived and reconstructed an ectopic pacing.
缓慢型心律失常是临床上常见的心律失常类型,严重影响患者的生活质量和生命安全。20世纪50年代,心脏植入性电子起搏装置的发明,为该病的治疗带来了革命性的变化。然而,心脏电子起搏器终究只是一种缓解症状的治疗方法,在长期的使用过程中也出现了诸多缺陷。心脏起搏生理的基础研究方面的进展,为起搏功能障碍的治疗开辟了新的前景,生物起搏器的概念也应运而生。近十余年以来,生物起搏器的研究取得了巨大进展。目前,生物起搏器的体内实验主要通过外科开胸手术的途径进行移植,存在创伤大、定位效果差等缺点,因此移植途径是制约生物起搏器临床应用的重要因素之一。探索适宜的移植手段是促进生物起搏进入临床实验阶段所需要克服的重要问题。本课题以犬的骨髓间充质干细胞(Bone-Marrow Mesenchymal Stem Cells, BM-MSCs)作为基因转染的靶细胞,构建超极化激活环核苷酸门控阳离子通道(Hyperpolarization-activated Cyclic Nucleotide-gated channels2, HCN2)基因的重组慢病毒载体,转染BM-MSCs,研究和验证转染后BM-MSCs在体外的起搏功能。采用白行研究设计的经皮心肌内注射导管,通过介入方法将体外构建的生物起搏器移植到实验动物犬的体内,探索该方法的安全性和可行性。
方法
采用密度梯度离心法分离犬的BM-MSCs并进行培养。扩增目的基因mHCN2,构建重组慢病毒表达载体。重组质粒和慢病毒包装系统共同转染T293细胞进行慢病毒的包装。Real-time PCR法检测病毒滴度。Lenti-GFP-HCN2以不同感染复数(multiplication of infection, MOI)转染第3-4代BM-MSCs,确定最佳MOI。设置Lenti-GFP-HCN2转染的BM-MSCs为实验组,Lenti-GFP转染的BM-MSCs为阴性对照组,未转染的BM-MSCs为空白对照组。全细胞膜片钳检测各组BM-MSCs的起搏电流。分离培养新生乳鼠心肌细胞(neonatal rat's cardiomyocytes, NRCs),将各组BM-MSCs和NRCs进行共培养,观察细胞搏动情况,并记录收缩频率。
经颈动脉和股静脉途径分别进入犬的左、右心室,进行龙胆紫稀释液的多点注射,检验自行研究设计的经皮心肌内注射导管的操作性能。将16只健康成年杂种犬(20-25kg)随机分为实验组(注射0.6m1转染了Lenti-GFP-HCN2的MSCs,n=8),阴性对照组(注射0.6m1转染了Lenti-GFP的MSCs, n=4)和空白对照组(注射0.6ml生理盐水,n=4)。经颈动脉途径放入自行设计的注射导管,在左心室室间隔位置定点注射。术后埋植体外遥测的心电记录仪实时监测犬的心率和心律。移植术后2周,对实验动物犬进行双侧迷走神经刺激(S1S1周长100ms/60ms,电压3-5V,时程1min,间隔10min)。记录逸搏心率的频率,分析逸搏来源以及迷走神经刺激后出现逸搏心率的时间间隔。术后2周达实验终点,留取心脏组织标本,冰冻切片后一部分直接在荧光显微镜下观察荧光,一部分进行HE染色以及Cx43,GFP和CD68的免疫荧光染色。
结果
基因测序表明目的基因HCN2已经成功重组至慢病毒表达载体,重组质粒转染T293细胞后,western blot证实在蛋白水平上有HCN2的过表达。本实验中确定慢病毒感染犬BM-MSCs的最佳MOI为20,在转染后48h开始表达绿色荧光,5天达高峰。实验组BM-MSCs可记录到超极化激活的内向电流,而阴性对照组和空白组BM-MSCs在相同记录条件下未能检测到明显的超极化内向电流。实验组BM-MSCs有HCN2蛋白过表达,而阴性对照组和空白组BM-MSCs未见表达。BM-MSCs和NRCs共培养后第3天,实验组NRCs的细胞收缩频率较阴性对照和空白对照组显著提高(P<0.05)。
经皮心肌内注射导管注射的有效性为91.6%,未出现严重并发症。移植术后各组的体外遥测心电记录仪显示室性早搏和室性心动过速的发生在各组间未见显著差异。双侧迷走神经刺激结果发现实验组的异搏心率主要来自注射腔左心室,频率为50.8±3.2bpm,而阴性对照组和空白组的异搏心率主要来自右心室,频率为42.2±6.1bpm(P<0.05)。免疫荧光检测显示介入方法移植到左心室的BM-MSCs在术后2周仍有存活,细胞之间有Cx43的表达和连接,HE染色和CD68的免疫荧光染色显示移植部位并没有显著炎症形成。
结论
mHCN2重组慢病毒(Lenti-GFP-HCN2)转染犬的BM-MSCs能在体外发挥起搏功能。自行研究设计的经皮心肌内注射导管可以用于在体移植转染的BM-MSCs,构建生物起搏器,该方法具有良好的有效性和安全性。利用该导管移植Lenti-GFP-HCN2转染的犬BM-MSCs可以在体内存活,并在移植部位重建起搏点,形成异位搏动。
Objective:
Bradyarrhythmia is a common arrhythmia type which severely affected the patient's health. The invitation of cardiac implanted electronic devices in1950s is a marvel, and advances in this field have been impressive. However, it's a relive therapy rather than cure, and important shortcomings exist. With the further development in cardiac pacing mechanism, the concept of biological pacemaker evolved, and the active investigation in the field of biological pacing is rapidly progressed. However, the in vivo animal studies of biological pacemaker are mainly performed through open thoracotomy, which cause large injury and had poor lacation controlling. Therefore, exploring optimal implantation approaches is very important for further clinic trails. This study used bone-marrow mesenchymal stem cells (BM-MSCs) as transfection target cells, and construct lentiviral expressing vectors carrying mouse Hyperpolarization-activated Cyclic Nucleotide-gated channels2(HCN2) gene. The in vitro pacing function of the transfected BM-MSCs was investigated. A self-designed percutaneous inject catheter was used to deliver the biological pacemaker into the left ventricular of canine, its feasibility and safety was explored. Meanwhile, the in vivo efficiency of the biological pacemaker was tested.
Methods:
Canine BM-MSCs was isolated by density gradient centrifugation and cultured. mHCN2gene fragment was amplified through PCR, then was connected with lentiviral vector. T293cells were con-transfected with lentiviral vector and packaging systems. The titers of lentiviral vectors were tested by real-time PCR. Gradient MOI of5,10,15,20,25,30was performed to determine the optimal MOI for the transfection of canine BM-MSCs. Set experiment group (Lenti-GFP-HCN2transfection), negative control group (Lenti-GFP transfection) and blank control group (with lentivirus transfection). Whole cell patch clamp tested the pacing current and western blot tested the HCN2protein expression of BM-MSCs in each group. NRCs were isolated and cultured, BM-MSCs was co-cultured with NRCs, the contraction frequency was observed and recorded. Catheter was inserted via carotid artery and femoral vein into left and right ventricle separately, diluted gentian violet was muitidot injected into the ventricle to testify the manipulation properties of the inject catheter.16health adult mongrel canine (20-25kg) were randomly divided into experiment group (0.6ml Lenti-GFP-HCN2 transfected MSCs was administrated, n=8), negative control group (0.6ml Lenti-GFP transfected MSCs was administrated, n=4), and blank control group (0.6ml saline was administrated, n=4). Inject catheter was inserted via carotid artery into left ventricle, septum inject was performed under the instruction of X-ray. Telemetering ECG recorder was implanted underneath the skin for heart rhythm monitoring.2weeks later, bilateral vague nerve stimulation was conducted (S1S1100ms/60ms, voltage3-5V, duration1min, interval10min). The escaping rhythm was recorded, its origin and frequency was analyzed. Heart tissue was obtained when end point was reached, frozen sections were sliced for GFP fluorescence, immunoflourescens stain and HE stain.
Results:
DNA sequencing demonstrated that mHCN2recombinated lentiviral vectors were constructed successfully. Western blot test confirmed that there was mHCN2protein expression in the T293cells, which was transfected with the mHCN2recombinated lentiviral vectors. The optimal lentivirus transfection MOI for canine BM-MSCs was20. Patch clamp showed the production of hyperpolarization-activated inward currents in experiment group while not in negative or blank control group. There was a high HCN2protein expression in experiment group other than negative and blank control groups.3days after co-culture, NRCs in experiment group revealed a significantly higher contraction frequency (P<0.05). The injection efficiency was calculated as91.6%, there was no severe complication associated with endomyocardial injection operation. Telemetering ECG recorder revealed premature ventricular contraction and ventricular tachycardia, and no significant differences were observed between three groups. Bilateral vague nerve stimulation showed that in experiment group, the escape rhythm mainly originated from left ventricle with an average frequency of50.8±3.2bpm, while the escape rhythm of negative and blank control groups from right ventricle, average frequency was42.2±6.1bpm (P<0.05). Histopathological examination proved that the injected BM-MSCs survived in vivo, there were Cx43connected between cells, HE stain and CD68immunoflourescens stain indicated no marked inflammation.
Conclusions:
In vitro transfection of canine BM-MSCs with Lenti-GFP-HCN2produced pacing current and expressed HCN2protein, indicating a potential of pacing function. The self-designed percutaneous endomyocardial inject catheter was proved to have highly-qualified efficiency and safety, it provided a better delivery for biological pacemaker study. Lenti-GFP-HCN2transfected canine BM-MSCs can be successfully delivered by this catheter, cells survived and reconstructed an ectopic pacing.
引文
1. Qu J, Barbuti A, Protas L, Santoro B, Cohen IS, Robinson RB. Hcn2 overexpression in newborn and adult ventricular myocytes:Distinct effects on gating and excitability. Circ Res.2001;89:E8-14
2. Miake J, Marban E, Nuss HB. Biological pacemaker created by gene transfer. Nature.2002;419:132-133
3. Ruhparwar A, Tebbenjohanns J, Niehaus M, Mengel M, Irtel T, Kofidis T, Pichlmaier AM, Haverich A. Transplanted fetal cardiomyocytes as cardiac pacemaker. Eur J Cardiothorac Surg.2002;21:853-857
4. Kehat I, Khimovich L, Caspi O, Gepstein A, Shofti R, Arbel G, Huber I, Satin J, Itskovitz-Eldor J, Gepstein L. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol.2004;22:1282-1289
5. Yamanaka S, Takahashi K. [induction of pluripotent stem cells from mouse fibroblast cultures]. Tanpakushitsu Kakusan Koso.2006;51:2346-2351
6. Takahashi K, Okita K, Nakagawa M, Yamanaka S. Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc.2007;2:3081-3089
7. Yoshida Y, Yamanaka S. Recent stem cell advances:Induced pluripotent stem cells for disease modeling and stem cell-based regeneration. Circulation. 2010;122:80-87
8. Okita K, Yamanaka S. Induced pluripotent stem cells: Opportunities and challenges. Philos Trans R Soc Lond B Biol Sci.2011;366:2198-2207
9. Clapham DE. Not so funny anymore:Pacing channels are cloned. Neuron. 1998;21:5-7
10. Baruscotti M, Barbuti A, Bucchi A. The cardiac pacemaker current. J Mol Cell Cardiol.2010;48:55-64
11. Mangoni ME, Nargeot J. Properties of the hyperpolarization-activated current (i(f)) in isolated mouse sino-atrial cells. Cardiovasc Res.2001;52:51-64
12. Verkerk AO, Wilders R, van Borren MM, Peters RJ, Broekhuis E, Lam K, Coronel R, de Bakker JM, Tan HL. Pacemaker current (i(f)) in the human sinoatrial node. Eur Heart J.2007;28:2472-2478
13. Bucchi A, Baruscotti M, Robinson RB, DiFrancesco D. Modulation of rate by autonomic agonists in san cells involves changes in diastolic depolarization and the pacemaker current. J Mol Cell Cardiol.2007;43:39-48
14. Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB, Rosen MR, Brink PR, Cohen IS. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol. 2004;555:617-626
15. Groh ME, Maitra B, Szekely E, Koc ON. Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive t cells. Exp Hematol. 2005;33:928-934
16. Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, Grisanti S, Gianni AM. Human bone marrow stromal cells suppress t-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood. 2002;99:3838-3843
17. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation.2002; 105:93-98
18. Qu J, Plotnikov AN, Danilo P, Jr., Shlapakova I, Cohen IS, Robinson RB, Rosen MR. Expression and function of a biological pacemaker in canine heart. Circulation.2003;107:1106-1109
19. Potapova I, Plotnikov A, Lu Z, Danilo P, Jr., Valiunas V, Qu J, Doronin S, Zuckerman J, Shlapakova IN, Gao J, Pan Z, Herron AJ, Robinson RB, Brink PR, Rosen MR, Cohen IS. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res.2004;94:952-959
20. Plotnikov AN, Shlapakova I, Szabolcs MJ, Danilo P, Jr., Lorell BH, Potapova I A, Lu Z, Rosen AB, Mathias RT, Brink PR, Robinson RB, Cohen IS, Rosen MR. Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart. Circulation. 2007;116:706-713
21. Plotnikov AN, Sosunov EA, Qu J, Shlapakova IN, Anyukhovsky EP, Liu L, Janse MJ, Brink PR, Cohen IS, Robinson RB, Danilo P, Jr., Rosen MR. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation. 2004;109:506-512
22. Bucchi A, Plotnikov AN, Shlapakova I, Danilo P, Jr., Kryukova Y, Qu J, Lu Z, Liu H, Pan Z, Potapova I, KenKnight B, Girouard S, Cohen IS, Brink PR, Robinson RB, Rosen MR. Wild-type and mutant hcn channels in a tandem biological-electronic cardiac pacemaker. Circulation.2006; 114:992-999
23. Macia E, Boyden PA. Stem cell therapy is proarrhythrnic. Circulation. 2009;119:1814-1823
24. Ly HQ, Nattel S. Stem cells are not proarrhythmic:Letting the genie out of the bottle. Circulation.2009;119:1824-1831
1. Furman S, Robinson G. Stimulation of the ventricular endocardial surface in control of complete heart block. Ann Surg.1959;150:841-845
2. Karlson KE, Gliedman ML. Cardiac pacemaker and defibrillator with variable output. J Thorac Surg.1959;37:208-213
3. Clark RM, Ross DN, Taylor DG, George RE. Complete heart-block; successful use of an electronic pacemaker after closure of ventricular septal defect. Lancet. 1959;1:392-394
4. Furman S, Schwedel JB. An intracardiac pacemaker for stokes-adams seizures. N Engl J Med.1959;261:943-948
5. Rosen MR, Brink PR, Cohen IS, Robinson RB. Cardiac pacing:From biological to electronic... To biological? Circ Arrhythm Electrophysiol.2008;1:54-61
6. Lee KL. In the wireless era:Leadless pacing. Expert Rev Cardiovasc Ther. 2010;8:171-174
7. Hauser RG, Hayes DL. Increasing hazard of sprint fidelis implantable cardioverter-defibrillator lead failure. Heart Rhythm.2009;6:605-610
8. Furrer M, Naegeli B, Bertel O. Hazards of an alternative medicine device in a patient with a pacemaker. N Engl J Med.2004;350:1688-1690
9. Martin ET, Coman JA, Shellock FG, Pulling CC, Fair R, Jenkins K. Magnetic resonance imaging and cardiac pacemaker safety at 1.5-tesla. J Am Coll Cardiol. 2004;43:1315-1324
10. Rosen MR, Brink PR, Cohen IS, Robinson RB. Genes, stem cells and biological pacemakers. Cardiovasc Res.2004;64:12-23
11. Siu CW, Lieu DK, Li RA. Hcn-encoded pacemaker channels:From physiology and biophysics to bioengineering. J Membr Biol.2006;214:115-122
12. Marban E, Cho HC. Biological pacemakers as a therapy for cardiac arrhythmias. Curr Opin Cardiol.2008;23:46-54
13. Biel M, Schneider A, Wahl C. Cardiac hcn channels:Structure, function, and modulation. Trends Cardiovasc Med.2002; 12:206-212
14. Bogdanov KY, Maltsev VA, Vinogradova TM, Lyashkov AE, Spurgeon HA, Stern MD, Lakatta EG. Membrane potential fluctuations resulting from submembrane ca2+ releases in rabbit sinoatrial nodal cells impart an exponential phase to the late diastolic depolarization that controls their chronotropic state. Circ Res. 2006;99:979-987
15. Zhang H, Holden AV, Boyett MR. Gradient model versus mosaic model of the sinoatrial node. Circulation.2001;103:584-588
16. Cohen IS, Robinson RB. Pacemaker current and automatic rhythms:Toward a molecular understanding. Handb Exp Pharmacol.2006:41-71
17. Edelberg JM, Huang DT, Josephson ME, Rosenberg RD. Molecular enhancement of porcine cardiac chronotropy. Heart.2001;86:559-562
18. Keating MT, Sanguinetti MC. Molecular and cellular mechanisms of cardiac arrhythmias. Cell.2001;104:569-580
19. Miake J, Marban E, Nuss HB. Functional role of inward rectifier current in heart probed by kir2.1 overexpression and dominant-negative suppression. J Clin Invest.2003;111:1529-1536
20. Zagotta WN, Olivier NB, Black KD, Young EC, Olson R, Gouaux E. Structural basis for modulation and agonist specificity of hen pacemaker channels. Nature. 2003;425:200-205
21. Altomare C, Bucchi A, Camatini E, Baruscotti M, Viscomi C, Moroni A, DiFrancesco D. Integrated allosteric model of voltage gating of hen channels. J Gen Physiol.2001;117:519-532
22. Accili EA, Proenza C, Baruscotti M, DiFrancesco D. From funny current to hen channels:20 years of excitation. News Physiol Sci.2002;17:32-37
23. Scicchitano P, Carbonara S, Ricci G, Mandurino C, Locorotondo M, Bulzis G, Gesualdo M, Zito A, Carbonara R, Dentamaro I, Riccioni G, Ciccone MM. Hen channels and heart rate. Molecules.2012;17:4225-4235
24. Taylor DA. Cell-based myocardial repair:How should we proceed? Int J Cardiol. 2004;95 Suppl 1:S8-12
25. Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells:Revisiting history, concepts, and assays. Cell Stem Cell.2008;2:313-319
26. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet.2004;364:149-155
27. Zannettino AC, Paton S, Arthur A, Khor F, Itescu S, Gimble JM, Gronthos S. Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo. J Cell Physiol.2008;214:413-421
28. Kim BO, Tian H, Prasongsukarn K, Wu J, Angoulvant D, Wnendt S, Muhs A, Spitkovsky D, Li RK. Cell transplantation improves ventricular function after a myocardial infarction:A preclinical study of human unrestricted somatic stem cells in a porcine model. Circulation.2005;112:196-104
29. Kuznetsov SA, Mankani MH, Gronthos S, Satomura K, Bianco P, Robey PG. Circulating skeletal stem cells. J Cell Biol.2001;153:1133-1140
30. Pountos I, Corscadden D, Emery P, Giannoudis PV. Mesenchymal stem cell tissue engineering: Techniques for isolation, expansion and application. Injury.2007;38 Suppl 4:S23-33
31. Tarnok A, Ulrich H, Bocsi J. Phenotypes of stem cells from diverse origin. Cytometry A.2010;77:6-10
32. Gaebel R, Furlani D, Sorg H, Polchow B, Frank J, Bieback K, Wang W, Klopsch C, Ong LL, Li W, Ma N, Steinhoff G. Cell origin of human mesenchymal stem cells determines a different healing performance in cardiac regeneration. PLoS One.2011;6:e15652
33. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science.1999;284:143-147
34. Ankrum J, Karp JM. Mesenchymal stem cell therapy: Two steps forward, one step back. Trends Mol Med.2010; 16:203-209
35. Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringden O. Hla expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol.2003;31:890-896
36. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood.2005;105:1815-1822
37. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol.2008;8:726-736
38. Nauta AJ, Westerhuis G, Kruisselbrink AB, Lurvink EG, Willemze R, Fibbe WE. Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood. 2006;108:2114-2120
1. Hamm A, Krott N, Breibach I, Blindt R, Bosserhoff AK. Efficient transfection method for primary cells. Tissue Eng.2002;8:235-245
2. Coonrod A, Li FQ, Horwitz M. On the mechanism of DNA transfection:Efficient gene transfer without viruses. Gene Ther.1997;4:1313-1321
3. Nakanishi M. New strategy in gene transfection by cationic transfection lipids with a cationic cholesterol. Curr Med Chem.2003;10:1289-1296
4. Pichon C, Billiet L, Midoux P. Chemical vectors for gene delivery:Uptake and intracellular trafficking. Curr Opin Biotechnol.2010;21:640-645
5. Gao X, Kim KS, Liu D. Nonviral gene delivery: What we know and what is next. AAPS J.2007;9:E92-104
6. Fiszer-Kierzkowska A, Vydra N, Wysocka-Wycisk A, Kronekova Z, Jarzab M, Lisowska KM, Krawczyk Z. Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells. BMC Mol Biol.2011;12:27
7. Kawabata K, Sakurai F, Koizumi N, Hayakawa T, Mizuguchi H. Adenovirus vector-mediated gene transfer into stem cells. Mol Pharm.2006;3:95-103
8. Potter H. Transfection by electroporation. Curr Protoc Immunol.2001;Chapter 10:Unit 10 15
9. Murakami T, Sunada Y. Plasmid DNA gene therapy by electroporation:Principles and recent advances. Curr Gene Ther.2011;11:447-456
10. Heilbronn R, Weger S. Viral vectors for gene transfer: Current status of gene therapeutics. Handb Exp Pharmacol.2010:143-170
11. Tannemaat MR, Verhaagen J, Malessy M. The application of viral vectors to enhance regeneration after peripheral nerve repair. Neurol Res. 2008;30:1039-1046
12. Matrai J, Chuah MK, VandenDriessche T. Recent advances in lentiviral vector development and applications. Mol Ther.2010; 18:477-490
13. Sakuma T, Barry MA, Ikeda Y. Lentiviral vectors:Basic to translational. Biochem J.2012;443:603-618
14. Dropulic B. Lentiviral vectors:Their molecular design, safety, and use in laboratory and preclinical research. Hum Gene Ther.2011;22:649-657
15. Qu J, Plotnikov AN, Danilo P, Jr., Shlapakova I, Cohen IS, Robinson RB, Rosen MR. Expression and function of a biological pacemaker in canine heart. Circulation.2003; 107:1106-1109
16. Plotnikov AN, Sosunov EA, Qu J, Shlapakova IN, Anyukhovsky EP, Liu L, Janse MJ, Brink PR, Cohen IS, Robinson RB, Danilo P, Jr., Rosen MR. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation. 2004;109:506-512
17. Shlapakova IN, Nearing BD, Lau DH, Boink GJ, Danilo P, Jr., Kryukova Y, Robinson RB, Cohen IS, Rosen MR, Verrier RL. Biological pacemakers in canines exhibit positive chronotropic response to emotional arousal. Heart Rhythm.2010;7:1835-1840
18. Pauwels K, Gijsbers R, Toelen J, Schambach A, Willard-Gallo K, Verheust C, Debyser Z, Herman P. State-of-the-art lentiviral vectors for research use:Risk assessment and biosafety recommendations. Curr Gene Ther.2009;9:459-474
19. Escors D, Breckpot K. Lentiviral vectors in gene therapy: Their current status and future potential. Arch Immunol Ther Exp (Warsz).2010;58:107-119
20. Pluta K, Kacprzak MM. Use of hiv as a gene transfer vector. Acta Biochim Pol. 2009;56:531-595
21. Ramezani A, Hawley RG. Overview of the hiv-1 lentiviral vector system. Curr Protoc Mol Biol.2002;Chapter 16:Unit 16 21
22. Barde I, Salmon P, Trono D. Production and titration of lentiviral vectors. Curr Protoc Neurosci.2010;Chapter 4:Unit 4 21
23. Lippincott-Schwartz J, Patterson GH. Development and use of fluorescent protein markers in living cells. Science.2003;300:87-91
24. Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB, Rosen MR, Brink PR, Cohen IS. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol. 2004;555:617-626
25. Valiunas V, Kanaporis G, Valiuniene L, Gordon C, Wang HZ, Li L, Robinson RB, Rosen MR, Cohen IS, Brink PR. Coupling an hcn2-expressing cell to a myocyte creates a two-cell pacing unit. J Physiol.2009;587:5211-5226
1. Rosen MR, Robinson RB, Brink PR, Cohen IS. The road to biological pacing. Nat Rev Cardiol.2011;8:656-666
2. Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB, Rosen MR, Brink PR, Cohen IS. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol. 2004;555:617-626
3. Valiunas V, Kanaporis G, Valiuniene L, Gordon C, Wang HZ, Li L, Robinson RB, Rosen MR, Cohen IS, Brink PR. Coupling an hcn2-expressing cell to a myocyte creates a two-cell pacing unit. J Physiol.2009;587:5211-5226
4. Jansen JA, van Veen TA, de Bakker JM, van Rijen HV. Cardiac connexins and impulse propagation. J Mol Cell Cardiol.2010;48:76-82
5. Plotnikov AN, Shlapakova I, Szabolcs MJ, Danilo P, Jr., Lorell BH, Potapova IA, Lu Z, Rosen AB, Mathias RT, Brink PR, Robinson RB, Cohen IS, Rosen MR. Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart. Circulation. 2007;116:706-713
6. Zhang H, Lau DH, Shlapakova IN, Zhao X, Danilo P, Robinson RB, Cohen IS, Qu D, Xu Z, Rosen MR. Implantation of sinoatrial node cells into canine right ventricle:Biological pacing appears limited by the substrate. Cell Transplant. 2011
7. Price MJ, Chou CC, Frantzen M, Miyamoto T, Kar S, Lee S, Shah PK, Martin BJ, Lill M, Forrester JS, Chen PS, Makkar RR. Intravenous mesenchymal stem cell therapy early after reperfused acute myocardial infarction improves left ventricular function and alters electrophysiologic properties. Int J Cardiol. 2006;111:231-239
8. Halkos ME, Zhao ZQ, Kerendi F, Wang NP, Jiang R, Schmarkey LS, Martin BJ, Quyyumi AA, Few WL, Kin H, Guyton RA, Vinten-Johansen J. Intravenous infusion of mesenchymal stem cells enhances regional perfusion and improves ventricular function in a porcine model of myocardial infarction. Basic Res Cardiol.2008; 103:525-536
9. Lipinski MJ, Biondi-Zoccai GG, Abbate A, Khianey R, Sheiban I, Bartunek J, Vanderheyden M, Kim HS, Kang HJ, Strauer BE, Vetrovec GW. Impact of intracoronary cell therapy on left ventricular function in the setting of acute myocardial infarction:A collaborative systematic review and meta-analysis of controlled clinical trials. J Am Coll Cardiol.2007;50:1761-1767
10. Zhang S, Sun A, Xu D, Yao K, Huang Z, Jin H, Wang K, Zou Y, Ge J. Impact of timing on efficacy and safetyof intracoronary autologous bone marrow stem cells transplantation in acute myocardial infarction:A pooled subgroup analysis of randomized controlled trials. Clin Cardiol.2009;32:458-466
11. Vulliet PR, Greeley M, Halloran SM, MacDonald KA, Kittleson MD. Intra-coronary arterial injection of mesenchymal stromal cells and microinfarction in dogs. Lancet.2004;363:783-784
12. Amado LC, Saliaris AP, Schuleri KH, St John M, Xie JS, Cattaneo S, Durand DJ, Fitton T, Kuang JQ, Stewart G, Lehrke S, Baumgartner WW, Martin BJ, Heldman AW, Hare JM. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A. 2005;102:11474-11479
13. Hou D, Youssef EA, Brinton TJ, Zhang P, Rogers P, Price ET, Yeung AC, Johnstone BH, Yock PG, March KL. Radiolabeled cell distribution after intramyocardial, intracoronary, and interstitial retrograde coronary venous delivery:Implications for current clinical trials. Circulation.2005;112:1150-156
14. Freyman T, Polin G, Osman H, Crary J, Lu M, Cheng L, Palasis M, Wilensky RL. A quantitative, randomized study evaluating three methods of mesenchymal stem cell delivery following myocardial infarction. Eur Heart J.2006;27:1114-1122
15. Yoon YS, Park JS, Tkebuchava T, Luedeman C, Losordo DW. Unexpected severe calcification after transplantation of bone marrow cells in acute myocardial infarction. Circulation.2004; 109:3154-3157
16. Perin EC, Silva GV, Assad JA, Vela D, Buja LM, Sousa AL, Litovsky S, Lin J, Vaughn WK, Coulter S, Fernandes MR, Willerson JT. Comparison of intracoronary and transendocardial delivery of allogeneic mesenchymal cells in a canine model of acute myocardial infarction. J Mol Cell Cardiol. 2008;44:486-495
17. Grossman PM, Han Z, Palasis M, Barry JJ, Lederman RJ. Incomplete retention after direct myocardial injection. Catheter Cardiovasc Interv.2002;55:392-397
18. Muller-Ehmsen J, Krausgrill B, Burst V, Schenk K, Neisen UC, Fries JW, Fleischmann BK, Hescheler J, Schwinger RH. Effective engraftment but poor mid-term persistence of mononuclear and mesenchymal bone marrow cells in acute and chronic rat myocardial infarction. J Mol Cell Cardiol.2006;41:876-884
19. Stamm C, Westphal B, Kleine HD, Petzsch M, Kittner C, Klinge H, Schumichen C, Nienaber CA, Freund M, Steinhoff G. Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet.2003;361:45-46
20. Kornowski R, Fuchs S, Tio FO, Pierre A, Epstein SE, Leon MB. Evaluation of the acute and chronic safety of the biosense injection catheter system in porcine hearts. Catheter Cardiovasc Interv.1999;48:447-453; discussion 454-445
21. Vale PR, Losordo DW, Tkebuchava T, Chen D, Milliken CE, Isner JM. Catheter-based myocardial gene transfer utilizing nonfluoroscopic electromechanical left ventricular mapping. J Am Coll Cardiol.1999;34:246-254
22. Kornowski R, Fuchs S, Leon MB, Epstein SE. Delivery strategies to achieve therapeutic myocardial angiogenesis. Circulation.2000; 101:454-458
23. Potapova I, Plotnikov A, Lu Z, Danilo P, Jr., Valiunas V, Qu J, Doronin S, Zuckerman J, Shlapakova IN, Gao J, Pan Z, Herron AJ, Robinson RB, Brink PR, Rosen MR, Cohen IS. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res.2004;94:952-959
1. Stieber J, Hofmann F, Ludwig A. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc Med.2004;14:23-28
2. 牛萍,黄从新,赵月强.重组起搏基因质粒构建生物起搏器的实验研究.中华心血管病杂志.2006：1126-1130
3. Barbuti A, DiFrancesco D. Control of cardiac rate by "funny" channels in health and disease. Ann N YAcad Sci.2008;1123:213-223
4. Clapham DE. Not so funny anymore:Pacing channels are cloned. Neuron. 1998;21:5-7
5. Baruscotti M, Barbuti A, Bucchi A. The cardiac pacemaker current. J Mol Cell Cardiol.2010;48:55-64
6. Mangoni ME, Nargeot J. Genesis and regulation of the heart automaticity. Physiol Rev.2008;88:919-982
7. DiFrancesco D, Tortora P. Direct activation of cardiac pacemaker channels by intracellular cyclic amp. Nature.1991;351:145-147
8. Accili EA, Proenza C, Baruscotti M, DiFrancesco D. From funny current to hen channels:20 years of excitation. News Physiol Sci.2002;17:32-37
9. Shi W, Wymore R, Yu H, Wu J, Wymore RT, Pan Z, Robinson RB, Dixon JE, McKinnon D, Cohen IS. Distribution and prevalence of hyperpolarization-activated cation channel (hen) mrna expression in cardiac tissues. Circ Res.1999;85:e1-6
10. Robinson RB, Siegelbaum SA. Hyperpolarization-activated cation currents:From molecules to physiological function. Annu Rev Physiol.2003;65:453-480
11. Biel M, Schneider A, Wahl C. Cardiac hcn channels:Structure, function, and modulation. Trends Cardiovasc Med.2002;12:206-212
12. Biel M, Wahl-Schott C, Michalakis S, Zong X. Hyperpolarization-activated cation channels:From genes to function. Physiol Rev.2009;89:847-885
13. Mangoni ME, Nargeot J. Properties of the hyperpolarization-activated current (i(f)) in isolated mouse sino-atrial cells. Cardiovasc Res.2001;52:51-64
14. Verkerk AO, Wilders R, van Borren MM, Peters RJ, Broekhuis E, Lam K, Coronel R, de Bakker JM, Tan HL. Pacemaker current (i(f)) in the human sinoatrial node. Eur Heart J.2007;28:2472-2478
15. Bucchi A, Baruscotti M, Robinson RB, DiFrancesco D. I(f)-dependent modulation of pacemaker rate mediated by camp in the presence of ryanodine in rabbit sino-atrial node cells. JMol Cell Cardiol.2003;35:905-913
16. Bucchi A, Baruscotti M, Robinson RB, DiFrancesco D. Modulation of rate by autonomic agonists in san cells involves changes in diastolic depolarization and the pacemaker current. J Mol Cell Cardiol.2007;43:39-48
17. Giorgetti A, Carloni P, Mistrik P, Torre V. A homology model of the pore region of hcn channels. Biophys J.2005;89:932-944
18. Schulze-Bahr E, Neu A, Friederich P, Kaupp UB, Breithardt G, Pongs O, Isbrandt D. Pacemaker channel dysfunction in a patient with sinus node disease. J Clin Invest.2003;111:1537-1545
19. Ueda K, Nakamura K, Hayashi T, Inagaki N, Takahashi M, Arimura T, Morita H, Higashiuesato Y, Hirano Y, Yasunami M, Takishita S, Yamashina A, Ohe T, Sunamori M, Hiraoka M, Kimura A. Functional characterization of a trafficking-defective hcn4 mutation, d553n, associated with cardiac arrhythmia. J Biol Chem.2004;279:27194-27198
20. Milanesi R, Baruscotti M, Gnecchi-Ruscone T, DiFrancesco D. Familial sinus bradycardia associated with a mutation in the cardiac pacemaker channel. N Engl J Med.2006;354:151-157
21. Nof E, Luria D, Brass D, Marek D, Lahat H, Reznik-Wolf H, Pras E, Dascal N, Eldar M, Glikson M. Point mutation in the hcn4 cardiac ion channel pore affecting synthesis, trafficking, and functional expression is associated with familial asymptomatic sinus bradycardia. Circulation.2007; 116:463-470
22. Laish-Farkash A, Glikson M, Brass D, Marek-Yagel D, Pras E, Dascal N, Antzelevitch C, Nof E, Reznik H, Eldar M, Luria D. A novel mutation in the hcn4 gene causes symptomatic sinus bradycardia in moroccan jews. J Cardiovasc Electrophysiol.2010;21:1365-1372
23. Qu J, Barbuti A, Protas L, Santoro B, Cohen IS, Robinson RB. Hcn2 overexpression in newborn and adult ventricular myocytes:Distinct effects on gating and excitability. Circ Res.2001;89:E8-14
24. Er F, Larbig R, Ludwig A, Biel M, Hofmann F, Beuckelmann DJ, Hoppe UC. Dominant-negative suppression of hen channels markedly reduces the native pacemaker current i(f) and undermines spontaneous beating of neonatal cardiomyocytes. Circulation.2003;107:485-489
25. 张翩,林国生,蔡军,马金安,曹庭家,曾彬,郭军.人超极化激活环核苷酸门控阳离子通道4基因体外转染乳鼠心肌细胞.中国心脏起搏与心电生理杂志.2009：23：47-50
26. 牛萍,黄从新,赵月强,杨波,赵庆彦,王腾,范国华.重组起搏基因质粒构建生物起搏器的实验研究.中华心血管病杂志.2006；34：1126-1130
27. Chan YC, Tse HF, Siu CW, Wang K, Li RA. Automaticity and conduction properties of bio-artificial pacemakers assessed in an in vitro monolayer model of neonatal rat ventricular myocytes. Europace.2010;12:1178-1187
28. Qu J, Plotnikov AN, Danilo P, Jr., Shlapakova I, Cohen IS, Robinson RB, Rosen MR. Expression and function of a biological pacemaker in canine heart. Circulation.2003;107:1106-1109
29. Plotnikov AN, Sosunov EA, Qu J, Shlapakova IN, Anyukhovsky EP, Liu L, Janse MJ, Brink PR, Cohen IS, Robinson RB, Danilo P, Jr., Rosen MR. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation. 2004;109:506-512
30. 张萍,郭继鸿,钟幼民,李继文,刘元伟,张幼怡,周春燕.人类超极化激活环核苷酸门控阳离子通道基因体内转染大鼠心脏进行生物起搏的初步研究.中华心血管病杂志.2007；35：275-276
31. 杨新春,易方方,蔡军,江洪,林国生,蒋学俊,杨波,胡莉华.腺病毒介导hcn4通道基因过度表达构建生物起搏器的实验研究.中华心血管病杂志.2007；35：277-279
32. Shlapakova IN, Nearing BD, Lau DH, Boink GJ, Danilo P, Jr., Kryukova Y, Robinson RB, Cohen IS, Rosen MR, Verrier RL. Biological pacemakers in canines exhibit positive chronotropic response to emotional arousal. Heart Rhythm.2010;7:1835-1840
33. Cho HC, Kashiwakura Y, Marban E. Creation of a biological pacemaker by cell fusion. Circ.Res.2007;100:1112-1115
34. Tse HF, Xue T, Lau CP, Siu CW, Wang K, Zhang QY, Tomaselli GF, Akar FG, Li RA. Bioartificial sinus node constructed via in vivo gene transfer of an engineered pacemaker hen channel reduces the dependence on electronic pacemaker in a sick-sinus syndrome model. Circulation.2006;114:1000-1011
35. Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB, Rosen MR, Brink PR, Cohen IS. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol. 2004;555:617-626
36. Groh ME, Maitra B, Szekely E, Koc ON. Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive t cells. Exp Hematol. 2005;33:928-934
37. Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, Grisanti S, Gianni AM. Human bone marrow stromal cells suppress t-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood. 2002;99:3838-3843
38. Plotnikov AN, Shlapakova I, Szabolcs MJ, Danilo P, Jr., Lorell BH, Potapova IA, Lu Z, Rosen AB, Mathias RT, Brink PR, Robinson RB, Cohen IS, Rosen MR. Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart. Circulation. 2007; 116:706-713
39. Potapova I, Plotnikov A, Lu Z, Danilo P, Jr., Valiunas V, Qu J, Doronin S, Zuckerman J, Shlapakova IN, Gao J, Pan Z, Herron AJ, Robinson RB, Brink PR, Rosen MR, Cohen IS. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res.2004;94:952-959
40. Yoon YS, Park JS, Tkebuchava T, Luedeman C, Losordo DW. Unexpected severe calcification after transplantation of bone marrow cells in acute myocardial infarction. Circulation.2004; 109:3154-3157
41. Breitbach M, Bostani T, Roell W, Xia Y, Dewald O, Nygren JM, Fries JW, Tiemann K, Bohlen H, Hescheler J, Welz A, Bloch W, Jacobsen SE, Fleischmann BK. Potential risks of bone marrow cell transplantation into infarcted hearts. Blood.2007;110:1362-1369
42. Zhang YM, Hartzell C, Narlow M, Dudley SC, Jr. Stem cell-derived cardiomyocytes demonstrate arrhythmic potential. Circulation. 2002; 106:1294-1299
43. Chang MG, Tung L, Sekar RB, Chang CY, Cysyk J, Dong P, Marban E, Abraham MR. Proarrhythmic potential of mesenchymal stem cell transplantation revealed in an in vitro coculture model. Circulation.2006;113:1832-1841
44. Cao JM, Fishbein MC, Han JB, Lai WW, Lai AC, Wu TJ, Czer L, Wolf PL, Denton TA, Shintaku IP, Chen PS, Chen LS. Relationship between regional cardiac hyperinnervation and ventricular arrhythmia. Circulation. 2000; 101:1960-1969
45. Burashnikov A, Antzelevitch C. Block of i(ks) does not induce early afterdepolarization activity but promotes beta-adrenergic agonist-induced delayed afterdepolarization activity. J Cardiovasc Electrophysiol.2000; 11:458-465
46. Pak HN, Qayyum M, Kim DT, Hamabe A, Miyauchi Y, Lill MC, Frantzen M, Takizawa K, Chen LS, Fishbein MC, Sharifi BG, Chen PS, Makkar R. Mesenchymal stem cell injection induces cardiac nerve sprouting and increased tenascin expression in a swine model of myocardial infarction. J Cardiovasc Electrophysiol.2003;14:841-848
2. Miake J, Marban E, Nuss HB. Biological pacemaker created by gene transfer. Nature.2002;419:132-133
3. Ruhparwar A, Tebbenjohanns J, Niehaus M, Mengel M, Irtel T, Kofidis T, Pichlmaier AM, Haverich A. Transplanted fetal cardiomyocytes as cardiac pacemaker. Eur J Cardiothorac Surg.2002;21:853-857
4. Kehat I, Khimovich L, Caspi O, Gepstein A, Shofti R, Arbel G, Huber I, Satin J, Itskovitz-Eldor J, Gepstein L. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol.2004;22:1282-1289
5. Yamanaka S, Takahashi K. [induction of pluripotent stem cells from mouse fibroblast cultures]. Tanpakushitsu Kakusan Koso.2006;51:2346-2351
6. Takahashi K, Okita K, Nakagawa M, Yamanaka S. Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc.2007;2:3081-3089
7. Yoshida Y, Yamanaka S. Recent stem cell advances:Induced pluripotent stem cells for disease modeling and stem cell-based regeneration. Circulation. 2010;122:80-87
8. Okita K, Yamanaka S. Induced pluripotent stem cells: Opportunities and challenges. Philos Trans R Soc Lond B Biol Sci.2011;366:2198-2207
9. Clapham DE. Not so funny anymore:Pacing channels are cloned. Neuron. 1998;21:5-7
10. Baruscotti M, Barbuti A, Bucchi A. The cardiac pacemaker current. J Mol Cell Cardiol.2010;48:55-64
11. Mangoni ME, Nargeot J. Properties of the hyperpolarization-activated current (i(f)) in isolated mouse sino-atrial cells. Cardiovasc Res.2001;52:51-64
12. Verkerk AO, Wilders R, van Borren MM, Peters RJ, Broekhuis E, Lam K, Coronel R, de Bakker JM, Tan HL. Pacemaker current (i(f)) in the human sinoatrial node. Eur Heart J.2007;28:2472-2478
13. Bucchi A, Baruscotti M, Robinson RB, DiFrancesco D. Modulation of rate by autonomic agonists in san cells involves changes in diastolic depolarization and the pacemaker current. J Mol Cell Cardiol.2007;43:39-48
14. Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB, Rosen MR, Brink PR, Cohen IS. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol. 2004;555:617-626
15. Groh ME, Maitra B, Szekely E, Koc ON. Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive t cells. Exp Hematol. 2005;33:928-934
16. Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, Grisanti S, Gianni AM. Human bone marrow stromal cells suppress t-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood. 2002;99:3838-3843
17. Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation.2002; 105:93-98
18. Qu J, Plotnikov AN, Danilo P, Jr., Shlapakova I, Cohen IS, Robinson RB, Rosen MR. Expression and function of a biological pacemaker in canine heart. Circulation.2003;107:1106-1109
19. Potapova I, Plotnikov A, Lu Z, Danilo P, Jr., Valiunas V, Qu J, Doronin S, Zuckerman J, Shlapakova IN, Gao J, Pan Z, Herron AJ, Robinson RB, Brink PR, Rosen MR, Cohen IS. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res.2004;94:952-959
20. Plotnikov AN, Shlapakova I, Szabolcs MJ, Danilo P, Jr., Lorell BH, Potapova I A, Lu Z, Rosen AB, Mathias RT, Brink PR, Robinson RB, Cohen IS, Rosen MR. Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart. Circulation. 2007;116:706-713
21. Plotnikov AN, Sosunov EA, Qu J, Shlapakova IN, Anyukhovsky EP, Liu L, Janse MJ, Brink PR, Cohen IS, Robinson RB, Danilo P, Jr., Rosen MR. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation. 2004;109:506-512
22. Bucchi A, Plotnikov AN, Shlapakova I, Danilo P, Jr., Kryukova Y, Qu J, Lu Z, Liu H, Pan Z, Potapova I, KenKnight B, Girouard S, Cohen IS, Brink PR, Robinson RB, Rosen MR. Wild-type and mutant hcn channels in a tandem biological-electronic cardiac pacemaker. Circulation.2006; 114:992-999
23. Macia E, Boyden PA. Stem cell therapy is proarrhythrnic. Circulation. 2009;119:1814-1823
24. Ly HQ, Nattel S. Stem cells are not proarrhythmic:Letting the genie out of the bottle. Circulation.2009;119:1824-1831
1. Furman S, Robinson G. Stimulation of the ventricular endocardial surface in control of complete heart block. Ann Surg.1959;150:841-845
2. Karlson KE, Gliedman ML. Cardiac pacemaker and defibrillator with variable output. J Thorac Surg.1959;37:208-213
3. Clark RM, Ross DN, Taylor DG, George RE. Complete heart-block; successful use of an electronic pacemaker after closure of ventricular septal defect. Lancet. 1959;1:392-394
4. Furman S, Schwedel JB. An intracardiac pacemaker for stokes-adams seizures. N Engl J Med.1959;261:943-948
5. Rosen MR, Brink PR, Cohen IS, Robinson RB. Cardiac pacing:From biological to electronic... To biological? Circ Arrhythm Electrophysiol.2008;1:54-61
6. Lee KL. In the wireless era:Leadless pacing. Expert Rev Cardiovasc Ther. 2010;8:171-174
7. Hauser RG, Hayes DL. Increasing hazard of sprint fidelis implantable cardioverter-defibrillator lead failure. Heart Rhythm.2009;6:605-610
8. Furrer M, Naegeli B, Bertel O. Hazards of an alternative medicine device in a patient with a pacemaker. N Engl J Med.2004;350:1688-1690
9. Martin ET, Coman JA, Shellock FG, Pulling CC, Fair R, Jenkins K. Magnetic resonance imaging and cardiac pacemaker safety at 1.5-tesla. J Am Coll Cardiol. 2004;43:1315-1324
10. Rosen MR, Brink PR, Cohen IS, Robinson RB. Genes, stem cells and biological pacemakers. Cardiovasc Res.2004;64:12-23
11. Siu CW, Lieu DK, Li RA. Hcn-encoded pacemaker channels:From physiology and biophysics to bioengineering. J Membr Biol.2006;214:115-122
12. Marban E, Cho HC. Biological pacemakers as a therapy for cardiac arrhythmias. Curr Opin Cardiol.2008;23:46-54
13. Biel M, Schneider A, Wahl C. Cardiac hcn channels:Structure, function, and modulation. Trends Cardiovasc Med.2002; 12:206-212
14. Bogdanov KY, Maltsev VA, Vinogradova TM, Lyashkov AE, Spurgeon HA, Stern MD, Lakatta EG. Membrane potential fluctuations resulting from submembrane ca2+ releases in rabbit sinoatrial nodal cells impart an exponential phase to the late diastolic depolarization that controls their chronotropic state. Circ Res. 2006;99:979-987
15. Zhang H, Holden AV, Boyett MR. Gradient model versus mosaic model of the sinoatrial node. Circulation.2001;103:584-588
16. Cohen IS, Robinson RB. Pacemaker current and automatic rhythms:Toward a molecular understanding. Handb Exp Pharmacol.2006:41-71
17. Edelberg JM, Huang DT, Josephson ME, Rosenberg RD. Molecular enhancement of porcine cardiac chronotropy. Heart.2001;86:559-562
18. Keating MT, Sanguinetti MC. Molecular and cellular mechanisms of cardiac arrhythmias. Cell.2001;104:569-580
19. Miake J, Marban E, Nuss HB. Functional role of inward rectifier current in heart probed by kir2.1 overexpression and dominant-negative suppression. J Clin Invest.2003;111:1529-1536
20. Zagotta WN, Olivier NB, Black KD, Young EC, Olson R, Gouaux E. Structural basis for modulation and agonist specificity of hen pacemaker channels. Nature. 2003;425:200-205
21. Altomare C, Bucchi A, Camatini E, Baruscotti M, Viscomi C, Moroni A, DiFrancesco D. Integrated allosteric model of voltage gating of hen channels. J Gen Physiol.2001;117:519-532
22. Accili EA, Proenza C, Baruscotti M, DiFrancesco D. From funny current to hen channels:20 years of excitation. News Physiol Sci.2002;17:32-37
23. Scicchitano P, Carbonara S, Ricci G, Mandurino C, Locorotondo M, Bulzis G, Gesualdo M, Zito A, Carbonara R, Dentamaro I, Riccioni G, Ciccone MM. Hen channels and heart rate. Molecules.2012;17:4225-4235
24. Taylor DA. Cell-based myocardial repair:How should we proceed? Int J Cardiol. 2004;95 Suppl 1:S8-12
25. Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells:Revisiting history, concepts, and assays. Cell Stem Cell.2008;2:313-319
26. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet.2004;364:149-155
27. Zannettino AC, Paton S, Arthur A, Khor F, Itescu S, Gimble JM, Gronthos S. Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo. J Cell Physiol.2008;214:413-421
28. Kim BO, Tian H, Prasongsukarn K, Wu J, Angoulvant D, Wnendt S, Muhs A, Spitkovsky D, Li RK. Cell transplantation improves ventricular function after a myocardial infarction:A preclinical study of human unrestricted somatic stem cells in a porcine model. Circulation.2005;112:196-104
29. Kuznetsov SA, Mankani MH, Gronthos S, Satomura K, Bianco P, Robey PG. Circulating skeletal stem cells. J Cell Biol.2001;153:1133-1140
30. Pountos I, Corscadden D, Emery P, Giannoudis PV. Mesenchymal stem cell tissue engineering: Techniques for isolation, expansion and application. Injury.2007;38 Suppl 4:S23-33
31. Tarnok A, Ulrich H, Bocsi J. Phenotypes of stem cells from diverse origin. Cytometry A.2010;77:6-10
32. Gaebel R, Furlani D, Sorg H, Polchow B, Frank J, Bieback K, Wang W, Klopsch C, Ong LL, Li W, Ma N, Steinhoff G. Cell origin of human mesenchymal stem cells determines a different healing performance in cardiac regeneration. PLoS One.2011;6:e15652
33. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science.1999;284:143-147
34. Ankrum J, Karp JM. Mesenchymal stem cell therapy: Two steps forward, one step back. Trends Mol Med.2010; 16:203-209
35. Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringden O. Hla expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol.2003;31:890-896
36. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood.2005;105:1815-1822
37. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol.2008;8:726-736
38. Nauta AJ, Westerhuis G, Kruisselbrink AB, Lurvink EG, Willemze R, Fibbe WE. Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood. 2006;108:2114-2120
1. Hamm A, Krott N, Breibach I, Blindt R, Bosserhoff AK. Efficient transfection method for primary cells. Tissue Eng.2002;8:235-245
2. Coonrod A, Li FQ, Horwitz M. On the mechanism of DNA transfection:Efficient gene transfer without viruses. Gene Ther.1997;4:1313-1321
3. Nakanishi M. New strategy in gene transfection by cationic transfection lipids with a cationic cholesterol. Curr Med Chem.2003;10:1289-1296
4. Pichon C, Billiet L, Midoux P. Chemical vectors for gene delivery:Uptake and intracellular trafficking. Curr Opin Biotechnol.2010;21:640-645
5. Gao X, Kim KS, Liu D. Nonviral gene delivery: What we know and what is next. AAPS J.2007;9:E92-104
6. Fiszer-Kierzkowska A, Vydra N, Wysocka-Wycisk A, Kronekova Z, Jarzab M, Lisowska KM, Krawczyk Z. Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells. BMC Mol Biol.2011;12:27
7. Kawabata K, Sakurai F, Koizumi N, Hayakawa T, Mizuguchi H. Adenovirus vector-mediated gene transfer into stem cells. Mol Pharm.2006;3:95-103
8. Potter H. Transfection by electroporation. Curr Protoc Immunol.2001;Chapter 10:Unit 10 15
9. Murakami T, Sunada Y. Plasmid DNA gene therapy by electroporation:Principles and recent advances. Curr Gene Ther.2011;11:447-456
10. Heilbronn R, Weger S. Viral vectors for gene transfer: Current status of gene therapeutics. Handb Exp Pharmacol.2010:143-170
11. Tannemaat MR, Verhaagen J, Malessy M. The application of viral vectors to enhance regeneration after peripheral nerve repair. Neurol Res. 2008;30:1039-1046
12. Matrai J, Chuah MK, VandenDriessche T. Recent advances in lentiviral vector development and applications. Mol Ther.2010; 18:477-490
13. Sakuma T, Barry MA, Ikeda Y. Lentiviral vectors:Basic to translational. Biochem J.2012;443:603-618
14. Dropulic B. Lentiviral vectors:Their molecular design, safety, and use in laboratory and preclinical research. Hum Gene Ther.2011;22:649-657
15. Qu J, Plotnikov AN, Danilo P, Jr., Shlapakova I, Cohen IS, Robinson RB, Rosen MR. Expression and function of a biological pacemaker in canine heart. Circulation.2003; 107:1106-1109
16. Plotnikov AN, Sosunov EA, Qu J, Shlapakova IN, Anyukhovsky EP, Liu L, Janse MJ, Brink PR, Cohen IS, Robinson RB, Danilo P, Jr., Rosen MR. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation. 2004;109:506-512
17. Shlapakova IN, Nearing BD, Lau DH, Boink GJ, Danilo P, Jr., Kryukova Y, Robinson RB, Cohen IS, Rosen MR, Verrier RL. Biological pacemakers in canines exhibit positive chronotropic response to emotional arousal. Heart Rhythm.2010;7:1835-1840
18. Pauwels K, Gijsbers R, Toelen J, Schambach A, Willard-Gallo K, Verheust C, Debyser Z, Herman P. State-of-the-art lentiviral vectors for research use:Risk assessment and biosafety recommendations. Curr Gene Ther.2009;9:459-474
19. Escors D, Breckpot K. Lentiviral vectors in gene therapy: Their current status and future potential. Arch Immunol Ther Exp (Warsz).2010;58:107-119
20. Pluta K, Kacprzak MM. Use of hiv as a gene transfer vector. Acta Biochim Pol. 2009;56:531-595
21. Ramezani A, Hawley RG. Overview of the hiv-1 lentiviral vector system. Curr Protoc Mol Biol.2002;Chapter 16:Unit 16 21
22. Barde I, Salmon P, Trono D. Production and titration of lentiviral vectors. Curr Protoc Neurosci.2010;Chapter 4:Unit 4 21
23. Lippincott-Schwartz J, Patterson GH. Development and use of fluorescent protein markers in living cells. Science.2003;300:87-91
24. Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB, Rosen MR, Brink PR, Cohen IS. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol. 2004;555:617-626
25. Valiunas V, Kanaporis G, Valiuniene L, Gordon C, Wang HZ, Li L, Robinson RB, Rosen MR, Cohen IS, Brink PR. Coupling an hcn2-expressing cell to a myocyte creates a two-cell pacing unit. J Physiol.2009;587:5211-5226
1. Rosen MR, Robinson RB, Brink PR, Cohen IS. The road to biological pacing. Nat Rev Cardiol.2011;8:656-666
2. Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB, Rosen MR, Brink PR, Cohen IS. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol. 2004;555:617-626
3. Valiunas V, Kanaporis G, Valiuniene L, Gordon C, Wang HZ, Li L, Robinson RB, Rosen MR, Cohen IS, Brink PR. Coupling an hcn2-expressing cell to a myocyte creates a two-cell pacing unit. J Physiol.2009;587:5211-5226
4. Jansen JA, van Veen TA, de Bakker JM, van Rijen HV. Cardiac connexins and impulse propagation. J Mol Cell Cardiol.2010;48:76-82
5. Plotnikov AN, Shlapakova I, Szabolcs MJ, Danilo P, Jr., Lorell BH, Potapova IA, Lu Z, Rosen AB, Mathias RT, Brink PR, Robinson RB, Cohen IS, Rosen MR. Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart. Circulation. 2007;116:706-713
6. Zhang H, Lau DH, Shlapakova IN, Zhao X, Danilo P, Robinson RB, Cohen IS, Qu D, Xu Z, Rosen MR. Implantation of sinoatrial node cells into canine right ventricle:Biological pacing appears limited by the substrate. Cell Transplant. 2011
7. Price MJ, Chou CC, Frantzen M, Miyamoto T, Kar S, Lee S, Shah PK, Martin BJ, Lill M, Forrester JS, Chen PS, Makkar RR. Intravenous mesenchymal stem cell therapy early after reperfused acute myocardial infarction improves left ventricular function and alters electrophysiologic properties. Int J Cardiol. 2006;111:231-239
8. Halkos ME, Zhao ZQ, Kerendi F, Wang NP, Jiang R, Schmarkey LS, Martin BJ, Quyyumi AA, Few WL, Kin H, Guyton RA, Vinten-Johansen J. Intravenous infusion of mesenchymal stem cells enhances regional perfusion and improves ventricular function in a porcine model of myocardial infarction. Basic Res Cardiol.2008; 103:525-536
9. Lipinski MJ, Biondi-Zoccai GG, Abbate A, Khianey R, Sheiban I, Bartunek J, Vanderheyden M, Kim HS, Kang HJ, Strauer BE, Vetrovec GW. Impact of intracoronary cell therapy on left ventricular function in the setting of acute myocardial infarction:A collaborative systematic review and meta-analysis of controlled clinical trials. J Am Coll Cardiol.2007;50:1761-1767
10. Zhang S, Sun A, Xu D, Yao K, Huang Z, Jin H, Wang K, Zou Y, Ge J. Impact of timing on efficacy and safetyof intracoronary autologous bone marrow stem cells transplantation in acute myocardial infarction:A pooled subgroup analysis of randomized controlled trials. Clin Cardiol.2009;32:458-466
11. Vulliet PR, Greeley M, Halloran SM, MacDonald KA, Kittleson MD. Intra-coronary arterial injection of mesenchymal stromal cells and microinfarction in dogs. Lancet.2004;363:783-784
12. Amado LC, Saliaris AP, Schuleri KH, St John M, Xie JS, Cattaneo S, Durand DJ, Fitton T, Kuang JQ, Stewart G, Lehrke S, Baumgartner WW, Martin BJ, Heldman AW, Hare JM. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci U S A. 2005;102:11474-11479
13. Hou D, Youssef EA, Brinton TJ, Zhang P, Rogers P, Price ET, Yeung AC, Johnstone BH, Yock PG, March KL. Radiolabeled cell distribution after intramyocardial, intracoronary, and interstitial retrograde coronary venous delivery:Implications for current clinical trials. Circulation.2005;112:1150-156
14. Freyman T, Polin G, Osman H, Crary J, Lu M, Cheng L, Palasis M, Wilensky RL. A quantitative, randomized study evaluating three methods of mesenchymal stem cell delivery following myocardial infarction. Eur Heart J.2006;27:1114-1122
15. Yoon YS, Park JS, Tkebuchava T, Luedeman C, Losordo DW. Unexpected severe calcification after transplantation of bone marrow cells in acute myocardial infarction. Circulation.2004; 109:3154-3157
16. Perin EC, Silva GV, Assad JA, Vela D, Buja LM, Sousa AL, Litovsky S, Lin J, Vaughn WK, Coulter S, Fernandes MR, Willerson JT. Comparison of intracoronary and transendocardial delivery of allogeneic mesenchymal cells in a canine model of acute myocardial infarction. J Mol Cell Cardiol. 2008;44:486-495
17. Grossman PM, Han Z, Palasis M, Barry JJ, Lederman RJ. Incomplete retention after direct myocardial injection. Catheter Cardiovasc Interv.2002;55:392-397
18. Muller-Ehmsen J, Krausgrill B, Burst V, Schenk K, Neisen UC, Fries JW, Fleischmann BK, Hescheler J, Schwinger RH. Effective engraftment but poor mid-term persistence of mononuclear and mesenchymal bone marrow cells in acute and chronic rat myocardial infarction. J Mol Cell Cardiol.2006;41:876-884
19. Stamm C, Westphal B, Kleine HD, Petzsch M, Kittner C, Klinge H, Schumichen C, Nienaber CA, Freund M, Steinhoff G. Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet.2003;361:45-46
20. Kornowski R, Fuchs S, Tio FO, Pierre A, Epstein SE, Leon MB. Evaluation of the acute and chronic safety of the biosense injection catheter system in porcine hearts. Catheter Cardiovasc Interv.1999;48:447-453; discussion 454-445
21. Vale PR, Losordo DW, Tkebuchava T, Chen D, Milliken CE, Isner JM. Catheter-based myocardial gene transfer utilizing nonfluoroscopic electromechanical left ventricular mapping. J Am Coll Cardiol.1999;34:246-254
22. Kornowski R, Fuchs S, Leon MB, Epstein SE. Delivery strategies to achieve therapeutic myocardial angiogenesis. Circulation.2000; 101:454-458
23. Potapova I, Plotnikov A, Lu Z, Danilo P, Jr., Valiunas V, Qu J, Doronin S, Zuckerman J, Shlapakova IN, Gao J, Pan Z, Herron AJ, Robinson RB, Brink PR, Rosen MR, Cohen IS. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res.2004;94:952-959
1. Stieber J, Hofmann F, Ludwig A. Pacemaker channels and sinus node arrhythmia. Trends Cardiovasc Med.2004;14:23-28
2. 牛萍,黄从新,赵月强.重组起搏基因质粒构建生物起搏器的实验研究.中华心血管病杂志.2006：1126-1130
3. Barbuti A, DiFrancesco D. Control of cardiac rate by "funny" channels in health and disease. Ann N YAcad Sci.2008;1123:213-223
4. Clapham DE. Not so funny anymore:Pacing channels are cloned. Neuron. 1998;21:5-7
5. Baruscotti M, Barbuti A, Bucchi A. The cardiac pacemaker current. J Mol Cell Cardiol.2010;48:55-64
6. Mangoni ME, Nargeot J. Genesis and regulation of the heart automaticity. Physiol Rev.2008;88:919-982
7. DiFrancesco D, Tortora P. Direct activation of cardiac pacemaker channels by intracellular cyclic amp. Nature.1991;351:145-147
8. Accili EA, Proenza C, Baruscotti M, DiFrancesco D. From funny current to hen channels:20 years of excitation. News Physiol Sci.2002;17:32-37
9. Shi W, Wymore R, Yu H, Wu J, Wymore RT, Pan Z, Robinson RB, Dixon JE, McKinnon D, Cohen IS. Distribution and prevalence of hyperpolarization-activated cation channel (hen) mrna expression in cardiac tissues. Circ Res.1999;85:e1-6
10. Robinson RB, Siegelbaum SA. Hyperpolarization-activated cation currents:From molecules to physiological function. Annu Rev Physiol.2003;65:453-480
11. Biel M, Schneider A, Wahl C. Cardiac hcn channels:Structure, function, and modulation. Trends Cardiovasc Med.2002;12:206-212
12. Biel M, Wahl-Schott C, Michalakis S, Zong X. Hyperpolarization-activated cation channels:From genes to function. Physiol Rev.2009;89:847-885
13. Mangoni ME, Nargeot J. Properties of the hyperpolarization-activated current (i(f)) in isolated mouse sino-atrial cells. Cardiovasc Res.2001;52:51-64
14. Verkerk AO, Wilders R, van Borren MM, Peters RJ, Broekhuis E, Lam K, Coronel R, de Bakker JM, Tan HL. Pacemaker current (i(f)) in the human sinoatrial node. Eur Heart J.2007;28:2472-2478
15. Bucchi A, Baruscotti M, Robinson RB, DiFrancesco D. I(f)-dependent modulation of pacemaker rate mediated by camp in the presence of ryanodine in rabbit sino-atrial node cells. JMol Cell Cardiol.2003;35:905-913
16. Bucchi A, Baruscotti M, Robinson RB, DiFrancesco D. Modulation of rate by autonomic agonists in san cells involves changes in diastolic depolarization and the pacemaker current. J Mol Cell Cardiol.2007;43:39-48
17. Giorgetti A, Carloni P, Mistrik P, Torre V. A homology model of the pore region of hcn channels. Biophys J.2005;89:932-944
18. Schulze-Bahr E, Neu A, Friederich P, Kaupp UB, Breithardt G, Pongs O, Isbrandt D. Pacemaker channel dysfunction in a patient with sinus node disease. J Clin Invest.2003;111:1537-1545
19. Ueda K, Nakamura K, Hayashi T, Inagaki N, Takahashi M, Arimura T, Morita H, Higashiuesato Y, Hirano Y, Yasunami M, Takishita S, Yamashina A, Ohe T, Sunamori M, Hiraoka M, Kimura A. Functional characterization of a trafficking-defective hcn4 mutation, d553n, associated with cardiac arrhythmia. J Biol Chem.2004;279:27194-27198
20. Milanesi R, Baruscotti M, Gnecchi-Ruscone T, DiFrancesco D. Familial sinus bradycardia associated with a mutation in the cardiac pacemaker channel. N Engl J Med.2006;354:151-157
21. Nof E, Luria D, Brass D, Marek D, Lahat H, Reznik-Wolf H, Pras E, Dascal N, Eldar M, Glikson M. Point mutation in the hcn4 cardiac ion channel pore affecting synthesis, trafficking, and functional expression is associated with familial asymptomatic sinus bradycardia. Circulation.2007; 116:463-470
22. Laish-Farkash A, Glikson M, Brass D, Marek-Yagel D, Pras E, Dascal N, Antzelevitch C, Nof E, Reznik H, Eldar M, Luria D. A novel mutation in the hcn4 gene causes symptomatic sinus bradycardia in moroccan jews. J Cardiovasc Electrophysiol.2010;21:1365-1372
23. Qu J, Barbuti A, Protas L, Santoro B, Cohen IS, Robinson RB. Hcn2 overexpression in newborn and adult ventricular myocytes:Distinct effects on gating and excitability. Circ Res.2001;89:E8-14
24. Er F, Larbig R, Ludwig A, Biel M, Hofmann F, Beuckelmann DJ, Hoppe UC. Dominant-negative suppression of hen channels markedly reduces the native pacemaker current i(f) and undermines spontaneous beating of neonatal cardiomyocytes. Circulation.2003;107:485-489
25. 张翩,林国生,蔡军,马金安,曹庭家,曾彬,郭军.人超极化激活环核苷酸门控阳离子通道4基因体外转染乳鼠心肌细胞.中国心脏起搏与心电生理杂志.2009：23：47-50
26. 牛萍,黄从新,赵月强,杨波,赵庆彦,王腾,范国华.重组起搏基因质粒构建生物起搏器的实验研究.中华心血管病杂志.2006；34：1126-1130
27. Chan YC, Tse HF, Siu CW, Wang K, Li RA. Automaticity and conduction properties of bio-artificial pacemakers assessed in an in vitro monolayer model of neonatal rat ventricular myocytes. Europace.2010;12:1178-1187
28. Qu J, Plotnikov AN, Danilo P, Jr., Shlapakova I, Cohen IS, Robinson RB, Rosen MR. Expression and function of a biological pacemaker in canine heart. Circulation.2003;107:1106-1109
29. Plotnikov AN, Sosunov EA, Qu J, Shlapakova IN, Anyukhovsky EP, Liu L, Janse MJ, Brink PR, Cohen IS, Robinson RB, Danilo P, Jr., Rosen MR. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation. 2004;109:506-512
30. 张萍,郭继鸿,钟幼民,李继文,刘元伟,张幼怡,周春燕.人类超极化激活环核苷酸门控阳离子通道基因体内转染大鼠心脏进行生物起搏的初步研究.中华心血管病杂志.2007；35：275-276
31. 杨新春,易方方,蔡军,江洪,林国生,蒋学俊,杨波,胡莉华.腺病毒介导hcn4通道基因过度表达构建生物起搏器的实验研究.中华心血管病杂志.2007；35：277-279
32. Shlapakova IN, Nearing BD, Lau DH, Boink GJ, Danilo P, Jr., Kryukova Y, Robinson RB, Cohen IS, Rosen MR, Verrier RL. Biological pacemakers in canines exhibit positive chronotropic response to emotional arousal. Heart Rhythm.2010;7:1835-1840
33. Cho HC, Kashiwakura Y, Marban E. Creation of a biological pacemaker by cell fusion. Circ.Res.2007;100:1112-1115
34. Tse HF, Xue T, Lau CP, Siu CW, Wang K, Zhang QY, Tomaselli GF, Akar FG, Li RA. Bioartificial sinus node constructed via in vivo gene transfer of an engineered pacemaker hen channel reduces the dependence on electronic pacemaker in a sick-sinus syndrome model. Circulation.2006;114:1000-1011
35. Valiunas V, Doronin S, Valiuniene L, Potapova I, Zuckerman J, Walcott B, Robinson RB, Rosen MR, Brink PR, Cohen IS. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol. 2004;555:617-626
36. Groh ME, Maitra B, Szekely E, Koc ON. Human mesenchymal stem cells require monocyte-mediated activation to suppress alloreactive t cells. Exp Hematol. 2005;33:928-934
37. Di Nicola M, Carlo-Stella C, Magni M, Milanesi M, Longoni PD, Matteucci P, Grisanti S, Gianni AM. Human bone marrow stromal cells suppress t-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood. 2002;99:3838-3843
38. Plotnikov AN, Shlapakova I, Szabolcs MJ, Danilo P, Jr., Lorell BH, Potapova IA, Lu Z, Rosen AB, Mathias RT, Brink PR, Robinson RB, Cohen IS, Rosen MR. Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart. Circulation. 2007; 116:706-713
39. Potapova I, Plotnikov A, Lu Z, Danilo P, Jr., Valiunas V, Qu J, Doronin S, Zuckerman J, Shlapakova IN, Gao J, Pan Z, Herron AJ, Robinson RB, Brink PR, Rosen MR, Cohen IS. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res.2004;94:952-959
40. Yoon YS, Park JS, Tkebuchava T, Luedeman C, Losordo DW. Unexpected severe calcification after transplantation of bone marrow cells in acute myocardial infarction. Circulation.2004; 109:3154-3157
41. Breitbach M, Bostani T, Roell W, Xia Y, Dewald O, Nygren JM, Fries JW, Tiemann K, Bohlen H, Hescheler J, Welz A, Bloch W, Jacobsen SE, Fleischmann BK. Potential risks of bone marrow cell transplantation into infarcted hearts. Blood.2007;110:1362-1369
42. Zhang YM, Hartzell C, Narlow M, Dudley SC, Jr. Stem cell-derived cardiomyocytes demonstrate arrhythmic potential. Circulation. 2002; 106:1294-1299
43. Chang MG, Tung L, Sekar RB, Chang CY, Cysyk J, Dong P, Marban E, Abraham MR. Proarrhythmic potential of mesenchymal stem cell transplantation revealed in an in vitro coculture model. Circulation.2006;113:1832-1841
44. Cao JM, Fishbein MC, Han JB, Lai WW, Lai AC, Wu TJ, Czer L, Wolf PL, Denton TA, Shintaku IP, Chen PS, Chen LS. Relationship between regional cardiac hyperinnervation and ventricular arrhythmia. Circulation. 2000; 101:1960-1969
45. Burashnikov A, Antzelevitch C. Block of i(ks) does not induce early afterdepolarization activity but promotes beta-adrenergic agonist-induced delayed afterdepolarization activity. J Cardiovasc Electrophysiol.2000; 11:458-465
46. Pak HN, Qayyum M, Kim DT, Hamabe A, Miyauchi Y, Lill MC, Frantzen M, Takizawa K, Chen LS, Fishbein MC, Sharifi BG, Chen PS, Makkar R. Mesenchymal stem cell injection induces cardiac nerve sprouting and increased tenascin expression in a swine model of myocardial infarction. J Cardiovasc Electrophysiol.2003;14:841-848