mHCN2基因修饰大鼠骨髓间充质干细胞用于构建生物起搏细胞的实验研究
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摘要
研究背景目前，治疗严重心动过缓的主要措施是植入电子起搏器。但是这种治疗方法仍存在一些缺陷与不足，如电池寿命有限、心脏永久性植入导管、不受神经体液因素调节等。为了进一步提高患者的生活质量，希望构建一种“生物起搏器”，使其可以象正常窦房结细胞一样在人体内表达起搏电流并通过缝隙连接传导，弥补窦房结或房室结功能不足，改善病人生存状况。
研究表明，超极化激活环核苷酸门控离子通道(HCN)表达的起搏电流在窦房结舒张期自动去极化过程中起着重要作用。HCN基因家族有4个成员，即HCN1～HCN4，其中HCN2对cAMP的反应强烈，有较快速的动力学等优点，是一种理想的生物起搏靶基因。干细胞具有自我增殖、多向分化潜能等优点，其研究及应用是国内外医学和生物学研究的热点之一。近年研究发现，成体干细胞不仅仅可以分化为组织特异性细胞，还具有跨系、跨胚层分化能力，同样可以分化为其他细胞或组织，为干细胞的应用开创了更为广泛的空间。此外，成体干细胞还具有易于取材，避免组织配型及免疫排斥反应，易于基因的导入及表达等优点。我们选用成体干细胞中的骨髓间充质干细胞作为传递基因的载体，利用转基因技术，将HCN2基因转入其中，构建“生物起搏器”，为将来替代电子起搏器用于临床治疗提供实验依据。
目的本研究以mHCN2为生物起搏的靶基因，应用大鼠的骨髓间充质干细胞(MSCs)作为生物起搏的平台，通过载体质粒转染获得mHCN2基因修饰的MSCs，并检测mHCN2基因在核酸、蛋白及电流水平的表达，为生物起搏技术的应用奠定试验基础并为其可行性提供依据。
方法采用密度梯度离心法和贴壁法相结合分离获得MSCs并用流式细胞术鉴定。EcoRⅠ和BamHⅠ双酶切质粒pGH-mHCN2和pIRES2-EGFP，回收目的片断，T4DNA连接酶连接。转化筛选阳性菌落，酶切和测序鉴定mHCN2是否插入pIRES2-EGFP中。脂质体转染质粒pIRES2-EGFP-mHCN2至MSCs，24～48h后，荧光显微镜下通过观察EGFP表达情况进一步判断转染效率。通过RT-PCR技术及Western blot方法检测已导入基因mHCN2的MSCs中mHCN2 mRNA和蛋白的表达情况。利用膜片钳技术分别记录转染和未转染质粒mHCN2的MSCs的内向电流，并记录Cs~+对电流的影响。
结果流式细胞仪检测结果显示获得纯度为95％的MSCs。酶切鉴定和测序结果均证明mHCN2片断插入质粒pIRES2-EGFP。荧光显微镜下可见转染了质粒的MSCs发出绿色荧光。已转染质粒MSCs的mHCN2 mRNA是未转染质粒MSCs的5．31倍(P＜0．05)，mHCN2蛋白是未转染的7．55倍(P＜0．05)。转染了mHCN2基因的细胞在超极化状态下记录到电压依赖型内向电流，未转染的MSCs未记录到该种电流。该电流在-140mV时被激活，阈电位为-60mV，半最大激活电位为(-95．1±0．9)mV。Cs+明显抑制该种电流。
结论成功构建真核表达质粒pIRES2-EGFP-mHCN2并通过脂质体转染方法使其在MSCs中表达。外源性mHCN2基因在MSCs中核酸和蛋白水平上均有表达，并且也成功表达了具有生理性起搏电流特征的I_(HCN2)。mHCN2基因修饰的MSCs有可能替代窦房结起搏细胞在自动除极过程中发挥重要作用。
Background Abnormalities of impulse generation and propagation induce cardiacarrhythmias. Although electronic pacemakers are currently the mainstay of therapy for heartblock and other electrophysiological abnormalities, they are not optimal. Among theirshortcomings are limited battery life, the need for permantent catherter implantation intothe heart, and lack of response to autonomic neurohumors. For these reasons, several genetherapy approaches have been explored as potential alternatives.
Hyperpolarization-activated cyclic nucleotide-gated channel (HCN) played animportant role in the automatic depolarization of the diastolic sinus node. HCN gene familyhave 4 members, HCN1-HCN4. HCN2 is an optimal biological pacemaker target genebecause it is essential for modulation funny current and maintenance of electric-physiologicfunction of pacemaker cells in sinus node. Self-proliferation and multipotency, two majoradvantages of stem cells, makes its research and application one of the hot spots in themedical and biological research activities at home and abroad. Where, embryonic stem cellsof the capacity to differentiate into all the tissues in the organism have drawn the mostattention. In spit of the tremendous potential in the medical applications, ethic controversiesarose due to the constraints its material sources. In recent years, studies found that morethan the capacities to differentiate into tissue-specific cells, adult stem cells can also have multi-lineage, multi-layer differentiation of embryos. In addition, they can differentiate intoother cells or tissues, creating broader applications of stem cells. Adult stem cells also canget easy access to material to avoid tissue typing and immunity rejection, easy for importand expression of gene. We selected rats mesenchymal stem cells, one kind of the adultstem cells, as the vector of genetic transmission, and tried to use transgenic technology toestablish the biological pacemaker, in attempt to replace electronic pacemakers in theapplication of clinical treatment.
Objective This experiment was executed using selected the rats mesenchymal stemcells modified by gene HCN2 as the target. The pacing current expressed by us constructthe plasmid pIRES2-EGFP-mHCN2 carrying a gene HCN2 and markers, and transferredthe plasmid to MSCs by liposome. Gene HCN2 was detected by the expression of nucleicacid, protein, and the current level, providing the evidences on the feasibility of testing forbiological pacemaker and mesenchymal stem cells as gene transfer vectors.
Methods MSCs were obtained by density gradient centrifugation method andadherence separation then identified by flow cytometer. The plasmid pGH-mHCN2 andplasmid pIRES2-EGFP were digested by EcoRⅠand BamHⅠ. The objective fragmentswere reclaimed and linked by T4 DNA Ligase. The recombinant plasmid was transformedto the competent cells and chose the masculine colony on the next day. Restriction enzymeand sequencing method were used to proof that mHCN2 was insert to pIRES2-EGFP. Theobjective gene was transfected with Lipofectamine 2000 into MSCs and the transfectingresults were observed by fluorescence microscope. The expression of mHCN2 mRNA andprotein in the transfected cells were identified by RT-PCR and Western blot. I_(HCN2) wasrecorded by whole-cell patch clamp. The effect of Cs~+ which is the specificity blocker ofpacemaker current on I_(HCN2) was detected.
Results MSCs were proved correctly by flow cytometer and the purity was 95%above. The identification using restriction enzyme and sequencing indicated that themHCN2 was inserted to the pIRES2-EGFP. The green fluorescence can be seen intransfected MSCs after 24 to 48 hours under fluorescence microscope. The mHCN2 mRNAin transfected MSCs is 5.31 times of the MSCs by RT-PCR (P＜0.05). The mHCN2 protein in transfected MSCs is 7.55 times of the MSCs by Western blot (P＜0.05). Non-transfectedMSCs demonstrated no significant voltage-dependent currents, mHCN2-transfected MSCsexpressed a large voltage-dependent inward current activating on hyperpolarizations. I_(HCN2)was fully activated around -140 mV with an activation threshold of -60 mV. The midpoint(V_(50)) was (-95.1±0.9) mV. Cs~+ (4 mmol/L) obviously blocked the current.
Conclusion We success construct the plasmid pIRES2-EGFP-mHCN2 and make itexpress in MSCs by Lipofectamine 2000.We demonstrate that mHCN2 gene can express inmRNA and protein levels in MSCs. mHCN2-transfected MSCs expressed the currents ofphysiological pacemaker current character. Its may substitute the sino-atrial nodepacemaker cells and play important effects in depolarization.
研究表明，超极化激活环核苷酸门控离子通道(HCN)表达的起搏电流在窦房结舒张期自动去极化过程中起着重要作用。HCN基因家族有4个成员，即HCN1～HCN4，其中HCN2对cAMP的反应强烈，有较快速的动力学等优点，是一种理想的生物起搏靶基因。干细胞具有自我增殖、多向分化潜能等优点，其研究及应用是国内外医学和生物学研究的热点之一。近年研究发现，成体干细胞不仅仅可以分化为组织特异性细胞，还具有跨系、跨胚层分化能力，同样可以分化为其他细胞或组织，为干细胞的应用开创了更为广泛的空间。此外，成体干细胞还具有易于取材，避免组织配型及免疫排斥反应，易于基因的导入及表达等优点。我们选用成体干细胞中的骨髓间充质干细胞作为传递基因的载体，利用转基因技术，将HCN2基因转入其中，构建“生物起搏器”，为将来替代电子起搏器用于临床治疗提供实验依据。
目的本研究以mHCN2为生物起搏的靶基因，应用大鼠的骨髓间充质干细胞(MSCs)作为生物起搏的平台，通过载体质粒转染获得mHCN2基因修饰的MSCs，并检测mHCN2基因在核酸、蛋白及电流水平的表达，为生物起搏技术的应用奠定试验基础并为其可行性提供依据。
方法采用密度梯度离心法和贴壁法相结合分离获得MSCs并用流式细胞术鉴定。EcoRⅠ和BamHⅠ双酶切质粒pGH-mHCN2和pIRES2-EGFP，回收目的片断，T4DNA连接酶连接。转化筛选阳性菌落，酶切和测序鉴定mHCN2是否插入pIRES2-EGFP中。脂质体转染质粒pIRES2-EGFP-mHCN2至MSCs，24～48h后，荧光显微镜下通过观察EGFP表达情况进一步判断转染效率。通过RT-PCR技术及Western blot方法检测已导入基因mHCN2的MSCs中mHCN2 mRNA和蛋白的表达情况。利用膜片钳技术分别记录转染和未转染质粒mHCN2的MSCs的内向电流，并记录Cs~+对电流的影响。
结果流式细胞仪检测结果显示获得纯度为95％的MSCs。酶切鉴定和测序结果均证明mHCN2片断插入质粒pIRES2-EGFP。荧光显微镜下可见转染了质粒的MSCs发出绿色荧光。已转染质粒MSCs的mHCN2 mRNA是未转染质粒MSCs的5．31倍(P＜0．05)，mHCN2蛋白是未转染的7．55倍(P＜0．05)。转染了mHCN2基因的细胞在超极化状态下记录到电压依赖型内向电流，未转染的MSCs未记录到该种电流。该电流在-140mV时被激活，阈电位为-60mV，半最大激活电位为(-95．1±0．9)mV。Cs+明显抑制该种电流。
结论成功构建真核表达质粒pIRES2-EGFP-mHCN2并通过脂质体转染方法使其在MSCs中表达。外源性mHCN2基因在MSCs中核酸和蛋白水平上均有表达，并且也成功表达了具有生理性起搏电流特征的I_(HCN2)。mHCN2基因修饰的MSCs有可能替代窦房结起搏细胞在自动除极过程中发挥重要作用。
Background Abnormalities of impulse generation and propagation induce cardiacarrhythmias. Although electronic pacemakers are currently the mainstay of therapy for heartblock and other electrophysiological abnormalities, they are not optimal. Among theirshortcomings are limited battery life, the need for permantent catherter implantation intothe heart, and lack of response to autonomic neurohumors. For these reasons, several genetherapy approaches have been explored as potential alternatives.
Hyperpolarization-activated cyclic nucleotide-gated channel (HCN) played animportant role in the automatic depolarization of the diastolic sinus node. HCN gene familyhave 4 members, HCN1-HCN4. HCN2 is an optimal biological pacemaker target genebecause it is essential for modulation funny current and maintenance of electric-physiologicfunction of pacemaker cells in sinus node. Self-proliferation and multipotency, two majoradvantages of stem cells, makes its research and application one of the hot spots in themedical and biological research activities at home and abroad. Where, embryonic stem cellsof the capacity to differentiate into all the tissues in the organism have drawn the mostattention. In spit of the tremendous potential in the medical applications, ethic controversiesarose due to the constraints its material sources. In recent years, studies found that morethan the capacities to differentiate into tissue-specific cells, adult stem cells can also have multi-lineage, multi-layer differentiation of embryos. In addition, they can differentiate intoother cells or tissues, creating broader applications of stem cells. Adult stem cells also canget easy access to material to avoid tissue typing and immunity rejection, easy for importand expression of gene. We selected rats mesenchymal stem cells, one kind of the adultstem cells, as the vector of genetic transmission, and tried to use transgenic technology toestablish the biological pacemaker, in attempt to replace electronic pacemakers in theapplication of clinical treatment.
Objective This experiment was executed using selected the rats mesenchymal stemcells modified by gene HCN2 as the target. The pacing current expressed by us constructthe plasmid pIRES2-EGFP-mHCN2 carrying a gene HCN2 and markers, and transferredthe plasmid to MSCs by liposome. Gene HCN2 was detected by the expression of nucleicacid, protein, and the current level, providing the evidences on the feasibility of testing forbiological pacemaker and mesenchymal stem cells as gene transfer vectors.
Methods MSCs were obtained by density gradient centrifugation method andadherence separation then identified by flow cytometer. The plasmid pGH-mHCN2 andplasmid pIRES2-EGFP were digested by EcoRⅠand BamHⅠ. The objective fragmentswere reclaimed and linked by T4 DNA Ligase. The recombinant plasmid was transformedto the competent cells and chose the masculine colony on the next day. Restriction enzymeand sequencing method were used to proof that mHCN2 was insert to pIRES2-EGFP. Theobjective gene was transfected with Lipofectamine 2000 into MSCs and the transfectingresults were observed by fluorescence microscope. The expression of mHCN2 mRNA andprotein in the transfected cells were identified by RT-PCR and Western blot. I_(HCN2) wasrecorded by whole-cell patch clamp. The effect of Cs~+ which is the specificity blocker ofpacemaker current on I_(HCN2) was detected.
Results MSCs were proved correctly by flow cytometer and the purity was 95%above. The identification using restriction enzyme and sequencing indicated that themHCN2 was inserted to the pIRES2-EGFP. The green fluorescence can be seen intransfected MSCs after 24 to 48 hours under fluorescence microscope. The mHCN2 mRNAin transfected MSCs is 5.31 times of the MSCs by RT-PCR (P＜0.05). The mHCN2 protein in transfected MSCs is 7.55 times of the MSCs by Western blot (P＜0.05). Non-transfectedMSCs demonstrated no significant voltage-dependent currents, mHCN2-transfected MSCsexpressed a large voltage-dependent inward current activating on hyperpolarizations. I_(HCN2)was fully activated around -140 mV with an activation threshold of -60 mV. The midpoint(V_(50)) was (-95.1±0.9) mV. Cs~+ (4 mmol/L) obviously blocked the current.
Conclusion We success construct the plasmid pIRES2-EGFP-mHCN2 and make itexpress in MSCs by Lipofectamine 2000.We demonstrate that mHCN2 gene can express inmRNA and protein levels in MSCs. mHCN2-transfected MSCs expressed the currents ofphysiological pacemaker current character. Its may substitute the sino-atrial nodepacemaker cells and play important effects in depolarization.
引文
1. DiFrancesco D. Funny channels in the control of cardiac rhythm and mode of action of selective blockers. Pharmacol Res, 2006, 53(5): 399-406
2. Moosmang Sven, Stieber Juliane, Zong Xiangang, et al. Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues. Eur J Biochem, 2001,268:1646-1652
3. Yerra L, Reddy PC. Effects of electromagnetic interference on implanted cardiac devices and their management. Cardiol Rev, 2007, 15(6): 304-309
4. Verkerk AO, Wilders R, van Borren MM, et al. Pacemaker current I(f) in the human sinoatrial node. Eur Heart J, 2007, 28(20): 2472-2478
5. DiFrancesco D, Borer JS. The funny current: cellular basis for the control of heart rate. Drugs, 2007, 67 (2): 15-24
6．冯凯．裴雪涛．间充质干细胞--现代组织工程的新资源．国外医学·生物医学工程分册．2000，23：325-329
7. Wislet-Gendebien S, Hans G, Leprince P, et al. Plasticity of cultured mesenchymal stem cells: switch from nestin-positive to excitable neuron-like phenotype. Stem Cells, 2005, 23(3): 392-402
8. Guillot P, Cook H, Pusey C, et al. Transplantation of human fetal mesenchymal stem cells improves glomerulopathy in a collagen type Ialpha2-deficient mouse. J Pathol, 2008, 214(5): 627-636
9. Liu J, Dobrzynski H, Yanni J, et al. Organisation of the mouse sinoatrial node: structure and expression of HCN channels. Cardiovasc Res, 2007, 73(4): 729-738
10. Rosen MR, Cohen IS, Brink PR, et al. Genes, stem cells and biological pace- makers. Cardiovascular Research, 2004, 64(1): 12-23
11. Plotnikov AN, Sosunov EA, Qu J, et al. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation, 2004, 109(4): 506-512
12. Bianco P, Riminucci M, Gronthos S, et al. Bone marrow stromal stem cells: nature, biology and potential applications. Stem Cells. 2001, 19(3): 180-192
13. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp. Hematol, 2000, 28(8): 875-884
14. Pittenger MF, Mackay AM, Beck SC, et al. Muitilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411):143-147
15. Martin DR, Cox NR, Hathcock TL, et al. Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow. Exp. Hematol, 2002, 30(8): 879-86
16. Devine SM, Bartholomew AM, Mahmud N, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primate following systemic infusion. Exp. Hematol, 2001, 29(2): 244-255
17. Ringe J, Kaps C, Schmitt B, et al. Porcine mesenchymal stem cells induction of distinct mesenchymal cell lineages. Cell tissue Res, 2002, 307(3): 321-327
18. Taiga Shibata, Keiko Naruse, Hideki Kamiya, et al. Transplantation of bone marrow-derived mesenchymal stem cells improves diabetic polyneuropathy in rats. Diabetes, 2008, 57:3099-3107
19. Friedenstein AJ, Chailakhyan RK, Gerasirnov UV. Bone marrow osteogenic stem cells: invitro cultivation and transplantation in diffusion chambers. Cell Tissuse Kinet, 1987, 20(3): 263-272
20. Deryugina EI, Muller-Sieburg CE. Stromal cells in longtem cultures: keys to the elucidation of hematopoietic development? Crit Rev Immunol, 1993, 13(2): 115-150
21．路艳蒙，傅文玉，朴英杰．大鼠间充质干细胞的培养．解剖学杂志，2000，23：160
22. Verkerk AO, Wilders R, van Borren MM, et al. Pacemaker current I(f) in the human sinoatrial node. Eur Heart J, 2007, 28(20): 2472-2478
23. Kawabata K, Sakurai F, Koizumi N, et al. Adenovirus vector-mediated gene transfer into stem cells. Mol Pharm, 2006, 3(2): 95-103
24. Zhou H, Ramiya VK, Visner GA. Bone marrow stem cells as a vehicle for delivery of heme oxygenase-1 gene. Stem Cells Dev, 2006, 15(1): 79-86
25. Aluigi M, Fogli M, Curti A, et al. Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mesenchymal stem cells. Stem Cells, 2006,24(2): 454-461
26. Takahashi T, Honmou O, Harada K, et al. Autoregulatory mechanism of Runx2 through the expression of transcription factors and bine matrix proteins in multipotential mesenchymal cell lines, ROB-C26. J Oral Sci, 2005, 47(4): 199-207
27. Nomura T, Honmou O, Harada K, et al. Infusion of brain-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat. Neuroscience, 2005, 136(1): 161-169
28. Byun HM, Suh D, Jeong Y, et al. Plasmid vectoes harboring cellular promoters can induce prolonged gene expression in hematopoietic and mesenchymal progenitor cells.Biochem Biophys Res Commum, 2005, 332(2): 518-523
29. Potapova I, Plotnikov A, Lu Z, et al. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res, 2004, 4(7): 952-959
30. Di Francesco D. A new interpretation of the pacemaker current IK2 in calf purkinje fibres. J Physiol, 1981, 314: 359-376
31. Santoro B, Grant SG, Bartsch D, et al. Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to Eag and cyclic nucleotide gated channels. Proc N atl Acad Sci USA, 1997, 94(26): 14815-14820
32. Wan Y. Involvement of hyperpolarization-activated, cyclic nucleotide-gated cation channels in dorsal root ganglion in neuropathic pain. Sheng Li Xue Bao, 2008, 60(5):579-580
33. Wahl-Schott C, Biel M. HCN channels: structure, cellular regulation and physiological function. Cell Mol Life Sci, 2009, 66(3): 470-494
34. Zhang Y, Zhang N, Gyulkhandanyan AV, et al. Presence of functional hyperpolarisation-activated cyclic nucleotide-gated channels in clonal alpha cell lines and rat islet alpha cells. Diabetologia, 2008, 51(12): 2290-2298
35. Yeh J, Kim BS, Gaines L, et al. The expression of hyperpolarization activated cyclic nucleotide gated (HCN) channels in the rat ovary are dependent on the type of cell and the reproductive age of the animal: a laboratory investigation. Reprod Biol Endocrinol, 2008, 6:35
36. Dibattista M, Mazzatenta A, Grassi F, et al. Hyperpolarization-activated cyclic nucleotide-gated channels in mouse vomeronasal sensory neurons. J Neurophysiol, 2008, 100(2): 576-586
37. Bolivar JJ, Tapia D, Arenas G, et al. A hyperpolarization-activated, cyclic nucleotide-gated, (Ih-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells. Am J Physiol Cell Physiol, 2008, 294(4): C893-906
38. Ludwig A, Zong X, Jeglitsch M, et al. A family of hyperpolarization-activated mammalian cation channels. Nature, 1998, 393(6685): 5872591
39. Robinson RB, Siegelbaum SA. Hyperpolarization-activated cation currents: from molecules to physiological function. Ann RevPhysiol, 2003, 65:453-480
40. Accili EA, Proenza C, Baruscotti M, et al. From fuuny current to HCN channels: 20 years of excitation. News Physiol. Sci, 2002, 17:32-37
41. Liu J, Dobrzynski H, Yanni J, et al. Organisation of the mouse sinoatrial node: structure and expression of HCN channels. Cardiovasc Res, 2007, 73(4): 729-738
42. Cheng L, Kinard K, Rajamani R, et al. Molecular Mapping of the Binding Site for a Blocker of Hyperpolarization-Activated, Cyclic Nucleotide-Modulated Pacemaker Channels. J. Pharmacol. Exp. Ther, 2007, 322(3): 931-939
43. Whitaker GM, Angoli D, Nazzari H, et al. HCN2 and HCN4 Isoforms Self-assemble and Co-assemble with Equal Preference to Form Functional Pacemaker Channels. J. Biol. Chem., 2007, 282(31): 22900 - 22909
44. Xue T, Siu CW, Lieu DK, et al. Mechanistic Role of If Revealed by Induction of Ventricular Automaticity by Somatic Gene Transfer of Gating-Engineered Pacemaker (HCN) Channels. Circulation, 2007, 115(14): 1839-1850
45. Shan C, Jing W, Steven AS, et al. Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCNl and HCN2 subunits and basalmuodulation by cyclic nucleotide. J Gen Physiol, 2001, 117(5): 491-503
46. Carlo V, Claudia A, Annalisa B, et al. C terminus-mediated control of voltage and cAMP gating of hyperpolarization-activated cyclic nucleotide-gated channels. J Biol Chem, 2001, 276(32): 29930-29934
47. Chen J, Piper DR, Sanguinetti MC, et al. Voltage sensing and activation gating of HCN pacemaker channels. Trends Cardiovasc Med, 2002, 419(6909): 837-841
48. Altomare C, Terragni B, Brioschi C. Heteromeric HCN1-HCN4 channels: a comparison with native pacemaker channels from the rabbit sinoatrial node. J Physiol, 2003,549(Pt2): 347-359
49. Zhang H, Dobrzynskl H, Kodama I, et al. How does the sinoatrial node drive the atrium? J Physiol, 2001, 536:69
50. Matsuura H, Ehara T, Fing WG, et al. Rapidly and slowly activating component of delayed rectifier K+ current in guinea-pig sinoatrial node pacemaker cells. J Physiol,2002, 540(3): 815-820
51. Kyoichi O, Shigehiro S, Toshihiko I, et al. Properties of the delayed rectifier potassium sinoatrial node cells. J Physiol, 2000, 524(1): 51-60
52. Stieber J, Herrmann S, Feil S, et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci USA, 2003,100(25):15235-15240
53. Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channel dysfunction in a patient with sinus node disease. J Clin Invest, 2003, 111(10): 1537-1545
54. Er F, Larbig R, Ludwig A, et al. Dominant-negative suppression of HCN channels markedly reduces the native pacemaker current If and undermines spontaneous baeting of neonatal cardiomyocytes. Circulation, 2003, 107:485-489
55. Ueda K, Nakamura K, Hayashi T, et al. Functional characerization of trafficking-defective HCN4 mutation, D553N, associated with cardiac arrhythmia. J Biol Chem, 2004, 279(26): 27194-27198
56. Milanesi R, Barruscoti M, DiFrancesco D, et al. Familial sinus bradycardia associated with a mutation in the cardiac pacemaker channel. N Engl Med, 2006, 353(2): 151-157
57. Zhang Z, Xu YF, Song HT, et al. Functional roles of Cav1.3(a, D) Calcium channel in sinoatrial nodes-insight gained using gene-targeted null mutant mice. Circ Res, 2002, 90:981-987
58. Koschak A, Reimer D, Huber L, et al. alD(Cav 1.3) subunits can from L-type Ca2+ channels activating at negative voltages. J Biol Chem, 2001,276:22100-22106
59. Honjo H, Inada S, Lancaster MK, et al. Sarcoplasmic reticulum Ca2+ release is not a dominating factor in sinoatrial node pacemaker activity at the diastolic potential range in rabbit sinoatrial node cells. Circ Res, 2003, 92:e41-e44
60. Martin Biel, Angela Schneider, Christian Wahl. Cardiac HCN channels, structure, function, and mudulation. Trends in cardiovascular medicine, 2002, 12(5): 206
61. Ruhparwar A, Tebbenjohanns J, Niehaus M, et al. Transplanted fetal cardiomyocytes as cardiac pacemaker. Criculation, 2001, 104(17): Ⅱ335
62. Ruhparwar A, Niehaus M, Radke K, et al. Enrichment of cells of the cardiac conduction system: Neuregulinl enhance the expression of connexin 40 in embryonic cardiomyocytes. Circulation, 2003, 108(17): Ⅳ240
63. Xiao Yong-Fu, Sigg Daniel C. Biological approaches to generating cardiac biopacemaker for bradycardia. Acta Physiologica Sinica, 2007, 59 (5): 562-570
64. Ohnishi S, Nagaya N. Prepare cells to repair the heart: mesenchymal stem cells for the treatment of heart failure. Am J Nephrol, 2007, 27(3): 301-307
65. Niemeyer P, Krause U, Kasten P, et al. Mesenchymal stem cell-based HLA-independent cell therapy for tissue engineering of bone and cartilage. Curr Stem Cell Res Ther, 2006,1(1): 21-27
66. Guo L, Kawazoe N, Fan Y, et al. Chondrogenic differentiation of human mesenchymal stem cells on photoreactive polymer-modified surfaces. Biomaterials, 2008, 29(1):23-32
67. DiFrancesco D, Borer JS. The funny current: cellular basis for the control of heart rate.Drugs, 2007, 67(2): 15-24
68. Kim HT, Kim IS, Lim SE, et al. Gene and cell replacement via neural stem cells. Yonsei Med J,2004, 45: 32-40
69. Fogle KJ, Lyashchenko AK, Turbendian HK, et al. HCN pacemaker channel activation is controlled by acidic lipids downstream of diacylglycerol kinase and phospholipase A2. JNeurosci, 2007, 27(11): 2802-2814.
70. Phillip Pian, Annalisa Bucchi, Richard B. Robinson, et al. Regulation of Gating and Rundown of HCN Hyperpolarization-activated Channels by Exogenous and Endogenous PIP2. The Rockefeller University Press, 2006, 128(5): 593-604
71. Lieu Deborah K, Chan Yau Chi, Lau Chu Pak, et al.Overexpression of HCN-encoded pacemaker current silences bioartificial pacemakers. Heart rhythm : the official journal of the Heart Rhythm Society, 2008, 5(9): 1310-1317
72. Akhavan A. Contribution of pacemaker channels to autonomous electrical activity of differentiated embryonic stem cells. J. Physiol, 2008, 586(10): 2425 - 2426
73. Bolivar JJ, Tapia D, Arenas G, et al. A hyperpolarization-activated, cyclic nucleotide-gated, (Ih-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells. Am J Physiol Cell Physiol, 2008, 294(4): C893 - C906
74. Qu Y, Whitakcr GM, L. Hove-Madsen, et al. Hyperpolarization-activated cyclic nucleotide-modulated 'HCN' channels confer regular and faster rhythmicity to beating mouse embryonic stem cells. J. Physiol., 2008, 586(3): 701 - 716
75. Baruscotti M, Robinson R. B. Electrophysiology and pacemaker function of the developing sinoatrial node. Am J Physiol Heart Circ Physiol, 2007, 293(5): H2613-H2623
76. El Chemaly A, Magaud C, Patri S, et al. The heart rate-lowering agent ivabradine inhibits the pacemaker current I(f) in human atrial myocytes. J Cardiovasc Electrophysiol, 2007, 18(11): 1190-1196
77. Siu CW, Lieu DK, Li RA. HCN-encoded pacemaker channels: from physiology and biophysics to bioengineering. J Membr Biol, 2006, 214(3): 115-122
78. Xue T, Siu CW, Lieu DK, et al. Mechanistic role of I(f) revealed by induction of ventricular automaticity by somatic gene transfer of gating-engineered pacemaker (HCN) channels. Circulation, 2007, 115(14): 1839-50
79. Zhou YF, Yang XJ, Li HX. Hyperpolarization-activated cyclic nucleotide-gated channel gene: the most possible therapeutic applications in the field of cardiac biological pacemakers. Med Hypotheses, 2007, 69(3):541-544
80. Yerra L, Reddy PC. Effects of electromagnetic interference on implanted cardiac devices and their management. Cardiol Rev, 2007, 15(6): 304-309
81. Eaton MJ. Cell and molecular approaches to the attenuation of pain after spinal cord injury. J Neurotrauma, 2006, 23(3-4):549-559
82. Mangoni ME, Nargeot J. Genesis and Regulation of the Heart Automaticity. Physiol Rev, 2008, 88(3): 919-982
1. Edelberg JM, Aird WC, Rosenberg RD. Enhancement of murine cardiac chronotropy by the molecular transfer of the human beta2 adrenergic receptor cDNA. J Clin Invest, 1998, 101(2): 337-343
2. Glenn CM, Pogwizd SM. Gene therapy to develop a genetically engineered cardiac pacemaker. J Cardiovasc Nurs, 2003, 18(5): 330-336
3. Qu J, Plotnikov AN, Danilo P Jr, et al. Expression and function of a biological pacemaker in canine heart. Circulation, 2003, 7(8): 1106-1109
4. Plotnikov AN, Sosunov EA, Qu J, et al. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation, 2004, 109(4): 506-512
5. Mujtaba T, Piper DR, Kalyani A, et al. Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells. Dev Biol, 1999, 214(1):113-127
6. Keller G, Wall C, Fong AZ, et al. Overexpression of H0X11 leads to the immortalization of embryonic precursors with both primitive and definitive hematopoietic potential. Blood, 1998, 92(3): 877-887
7. Noboru Sato, Laurent Meijer, Leandros Skaltsounis, et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nature medicine, 2004, 10:55-63
8. Lisa M Hoffman, Melissa K Carpenter, et al. Characterization and culture of human embryonic stem cells. Nature biotechnology, 2005, 23:699-708
9. Yasushi Takagi, Jun Takahashi, Hidemoto Saiki, et al. Dopaminergic neurons generated from monkey embryonic stem cells function in a parkinson primate model. J. Clin.Invest, 2005, 115(1): 102-109
10. Przemyslaw Blyszczuk, Jaroslaw Czyz, Gabriela Kania, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. PNAS, 2003, 100(3): 998-1003
11. Thomas P. Zwaka, James A. Thomson. Homologous recombination in human embryonic stem cells. Nature biotechnology, 2003, 21: 319-321
12. Chad A. Cowan, Irina Klimanskaya, Jill McMahon, et al. Derivation of Embryonic Stem-Cell Lines from Human Blastocysts. The new England Journal of medine, 2004,350(13): 1353-1356
13. Lisa M Hoffman & Melissa K Carpenter.Characterization and culture of human embryonic stem cells. Nrture Biotechnology, 2005, 23:699-708
14. Assady S, Maor G, Amit M, et al. Insulin production by human embryonic stem cells. Diabetes, 2001, 50(8): 1691-1697
15. Fijnvandraat AC, De Boer PA, Deprez RH, et al. Non- radioactive in situ detection of mRNA in ES cell-derived cardiomyocytes and in the developing heart. Microsc Res Tech, 2002, 58(5): 387-394
16. Kawasaki H, Suemori H, Mizuseki K, et al. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci U S A,2002, 9(3): 1580-1585
17. Ruhparwar A, Tebbenjohanns J, Niehaus M, et al. Transplanted fetal cardiomyocytes as cardiac pacemaker. Eur J Cardiothorac Surg, 2002, (5): 53-57
18. Ruhparwar A, Haverich A. Prospects for iological cardiac pacemaker systems. Pacing Clin Electrophysiol, 2003, (11): 2069-207
19. Kehat I, Khimovich L, Caspi O, et al. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol, 2004 (10): 1282-1289
20. Tian Xue, Hee Cheol Cho, Fadi G. Akar, et al. Functional Integration of Electrically Active Cardiac Derivatives From Genetically Engineered Human Embryonic Stem Cells With Quiescent Recipient Ventricular. Circulation, 2005, 112(6): e82- e83
21. Ohnishi S, Nagaya N. Prepare cells to repair the heart: mesenchymal stem cells for the treatment of heart failure. Am JNephrol, 2007, 27(3): 301-307
22. Niemeyer P, Krause U, Kasten P, et al. Mesenchymal stem cell-based HLA-independent cell therapy for tissue engineering of bone and cartilage. Curr Stem Cell Res Ther,2006, 1(1): 21-27
23. Guo L, Kawazoe N, Fan Y, et al. Chondrogenic differentiation of human mesenchymal stem cells on photoreactive polymer-modified surfaces. Biomaterials, 2008, 29(1):23-32
24. Valiunas V, Doronin S, Valiuniene L, et al. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol, 2004, (55): 617-626
25. Potapova I, Plotnikov A, Lu Z, et al. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res, 2004, 4(7): 952-959
26. Beeres SL, Atsma DE, van der Laarse A,et al.Human adult bone marrow mesenchymal stem cells repair experimental conduction block in rat cardiomyocyte cultures. J Am Coll Cardiol, 2005, 6(10): 1943-1952
27. Alexei N. Plotnikov, Eugene A. Sosunov, et al. Biological Pacemaker Implanted in Canine Left Bundle Branch Provides Ventricular Escape Rhythms That Have Physiologically Acceptable Rates. Circulation, 2004, 5(9): 506-512
28. Plotnikov AN, Sosunov EA, Qu J, et al. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation, 2004, (4): 506-512
29. Stieber J, Hofmann F, Ludwig A. Pacemaker channels and sinus node arrhythmia.Trends Cardiovasc Med, 2004, (41): 23-28
30. Ueda K, Nakamura K, Hayashi T, et al. Functional charac- terization of a trafficking-defective HCN4 mutation, D553N, associatedu with cardiac arrhythmia. J Biol Chem, 2004, 79(26): 27194-27198
31. Stieber J, Herrmann S, Feil S, et al. The hyperpolarization- activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci U S A, 2003, 100(25): 15235-15240.
32. Garcia-Frigola C, Shi Y, et al. Expression of the hyperpo- larization-activated cyclic nucleotide-gated cation channel HCN4 during mouse heart development. Gene Expr Patterns, 2003, (6): 777-783
33. Zicha S, Fernandez-Velasco M, Lonardo G, et al. Sinus node dysfunction and hyperpolarization-activated (HCN) channel subunit remodeling in a canine heart failure model. Cardiovasc Res, 2005, 6(3): 430-432
34. Mark F. Pittenger, Alastair M. Mackay, Stephen C. Beck, et al. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science, 1999, 284(5411): 143-147
35. James A. Thomson, Joseph Itskovitz-Eldor, Sander S., et al. Embryonic Stem Cell Lines Derived from Human Blastocysts. Science, 1998, 282(5391): 1145 - 1147
36. Eric Lagasse, Heather Connors. Muhsen Al-Dhalimy, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo .Nature Medicine, 2000, 6: 1229 - 1234
37. Fred H. Gage. Mammalian Neural Stem Cells. Science, 2000, 287(5457): 1433-1438
38. Imhof Alexander, Balajee S. Arunmozhi, Fredricks David N., et al. Breakthrough Fungal Infections in Stem Cell Transplant Recipients Receiving Voriconazole. Clinical Infectious Diseases, 2004, 39: 743-746
39. Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood, 1993, 81(11): 2844-2853
40. Luciano C. Amado, Anastasios P. Saliaris, Karl H. Schuleri, et al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. PNAS, 2005, 102(32): 11474-11479
41. Andreas Sch(a|¨)ffler, Christa B(u|¨)chler, et al.Concise Review: Adipose Tissue-Derived Stromal Cells - Basic and Clinical Implications for Novel Cell-Based Therapies. Stem cells, 2007, 25(4): 818-827
42. Massimiliano Gnecchi, Huamei He, Olin D Liang, et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nature medine,2005, 11:367-368
43. Lakshmipathy U, Verfaillie C. Stem cell plasticity. Blood Reviews, 19(1) :29-38
44. Catalin Toma, Mark F. Pittenger, Kevin S. Cahill, et al. Human Mesenchymal Stem Cells Differentiate to a Cardiomyocyte Phenotype in the Adult Murine Heart.Circulation, 2002, 105:93
45. Mark F. Pittenger, Alastair M. Mackay, Stephen C. Beck, et al. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science,1999, 284(5411):143-147
46. Siu CW, Lieu DK, Li RA. HCN-encoded pacemaker channels: from physiology and biophysics to bioengineering. J Membr Biol, 2006, 214(3): 115-122
47. Xue T, Siu CW, Lieu DK, et al. Mechanistic role of I(f) revealed by induction of ventricular automaticity by somatic gene transfer of gating-engineered pacemaker (HCN) channels. Circulation, 2007, 115(14): 1839-50
48. Zhou YF, Yang XJ, Li HX. Hyperpolarization-activated cyclic nucleotide-gated channel gene: the most possible therapeutic applications in the field of cardiac biological pacemakers. Med Hypotheses, 2007, 69(3): 541-544
49. Yerra L, Reddy PC. Effects of electromagnetic interference on implanted cardiac devices and their management. Cardiol Rev, 2007, 15(6): 304-309
50. Verkerk AO, Wilders R, van Borren MM, et al.Pacemaker current I(f) in the human sinoatrial node. Eur Heart J, 2007, 28(20):2472-2478
51. DiFrancesco D, Borer JS. The funny current: cellular basis for the control of heart rate.Drugs, 2007, 67(2): 15-24
52. Xiao Yong-Fu, Daniel C. Sigg. Biological approaches to generating cardiac biopacemaker for bradycardia . Acta Physiologica Sinica, 2007, 59 (5): 562-570
2. Moosmang Sven, Stieber Juliane, Zong Xiangang, et al. Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues. Eur J Biochem, 2001,268:1646-1652
3. Yerra L, Reddy PC. Effects of electromagnetic interference on implanted cardiac devices and their management. Cardiol Rev, 2007, 15(6): 304-309
4. Verkerk AO, Wilders R, van Borren MM, et al. Pacemaker current I(f) in the human sinoatrial node. Eur Heart J, 2007, 28(20): 2472-2478
5. DiFrancesco D, Borer JS. The funny current: cellular basis for the control of heart rate. Drugs, 2007, 67 (2): 15-24
6．冯凯．裴雪涛．间充质干细胞--现代组织工程的新资源．国外医学·生物医学工程分册．2000，23：325-329
7. Wislet-Gendebien S, Hans G, Leprince P, et al. Plasticity of cultured mesenchymal stem cells: switch from nestin-positive to excitable neuron-like phenotype. Stem Cells, 2005, 23(3): 392-402
8. Guillot P, Cook H, Pusey C, et al. Transplantation of human fetal mesenchymal stem cells improves glomerulopathy in a collagen type Ialpha2-deficient mouse. J Pathol, 2008, 214(5): 627-636
9. Liu J, Dobrzynski H, Yanni J, et al. Organisation of the mouse sinoatrial node: structure and expression of HCN channels. Cardiovasc Res, 2007, 73(4): 729-738
10. Rosen MR, Cohen IS, Brink PR, et al. Genes, stem cells and biological pace- makers. Cardiovascular Research, 2004, 64(1): 12-23
11. Plotnikov AN, Sosunov EA, Qu J, et al. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation, 2004, 109(4): 506-512
12. Bianco P, Riminucci M, Gronthos S, et al. Bone marrow stromal stem cells: nature, biology and potential applications. Stem Cells. 2001, 19(3): 180-192
13. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp. Hematol, 2000, 28(8): 875-884
14. Pittenger MF, Mackay AM, Beck SC, et al. Muitilineage potential of adult human mesenchymal stem cells. Science, 1999, 284(5411):143-147
15. Martin DR, Cox NR, Hathcock TL, et al. Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow. Exp. Hematol, 2002, 30(8): 879-86
16. Devine SM, Bartholomew AM, Mahmud N, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primate following systemic infusion. Exp. Hematol, 2001, 29(2): 244-255
17. Ringe J, Kaps C, Schmitt B, et al. Porcine mesenchymal stem cells induction of distinct mesenchymal cell lineages. Cell tissue Res, 2002, 307(3): 321-327
18. Taiga Shibata, Keiko Naruse, Hideki Kamiya, et al. Transplantation of bone marrow-derived mesenchymal stem cells improves diabetic polyneuropathy in rats. Diabetes, 2008, 57:3099-3107
19. Friedenstein AJ, Chailakhyan RK, Gerasirnov UV. Bone marrow osteogenic stem cells: invitro cultivation and transplantation in diffusion chambers. Cell Tissuse Kinet, 1987, 20(3): 263-272
20. Deryugina EI, Muller-Sieburg CE. Stromal cells in longtem cultures: keys to the elucidation of hematopoietic development? Crit Rev Immunol, 1993, 13(2): 115-150
21．路艳蒙，傅文玉，朴英杰．大鼠间充质干细胞的培养．解剖学杂志，2000，23：160
22. Verkerk AO, Wilders R, van Borren MM, et al. Pacemaker current I(f) in the human sinoatrial node. Eur Heart J, 2007, 28(20): 2472-2478
23. Kawabata K, Sakurai F, Koizumi N, et al. Adenovirus vector-mediated gene transfer into stem cells. Mol Pharm, 2006, 3(2): 95-103
24. Zhou H, Ramiya VK, Visner GA. Bone marrow stem cells as a vehicle for delivery of heme oxygenase-1 gene. Stem Cells Dev, 2006, 15(1): 79-86
25. Aluigi M, Fogli M, Curti A, et al. Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mesenchymal stem cells. Stem Cells, 2006,24(2): 454-461
26. Takahashi T, Honmou O, Harada K, et al. Autoregulatory mechanism of Runx2 through the expression of transcription factors and bine matrix proteins in multipotential mesenchymal cell lines, ROB-C26. J Oral Sci, 2005, 47(4): 199-207
27. Nomura T, Honmou O, Harada K, et al. Infusion of brain-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat. Neuroscience, 2005, 136(1): 161-169
28. Byun HM, Suh D, Jeong Y, et al. Plasmid vectoes harboring cellular promoters can induce prolonged gene expression in hematopoietic and mesenchymal progenitor cells.Biochem Biophys Res Commum, 2005, 332(2): 518-523
29. Potapova I, Plotnikov A, Lu Z, et al. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res, 2004, 4(7): 952-959
30. Di Francesco D. A new interpretation of the pacemaker current IK2 in calf purkinje fibres. J Physiol, 1981, 314: 359-376
31. Santoro B, Grant SG, Bartsch D, et al. Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to Eag and cyclic nucleotide gated channels. Proc N atl Acad Sci USA, 1997, 94(26): 14815-14820
32. Wan Y. Involvement of hyperpolarization-activated, cyclic nucleotide-gated cation channels in dorsal root ganglion in neuropathic pain. Sheng Li Xue Bao, 2008, 60(5):579-580
33. Wahl-Schott C, Biel M. HCN channels: structure, cellular regulation and physiological function. Cell Mol Life Sci, 2009, 66(3): 470-494
34. Zhang Y, Zhang N, Gyulkhandanyan AV, et al. Presence of functional hyperpolarisation-activated cyclic nucleotide-gated channels in clonal alpha cell lines and rat islet alpha cells. Diabetologia, 2008, 51(12): 2290-2298
35. Yeh J, Kim BS, Gaines L, et al. The expression of hyperpolarization activated cyclic nucleotide gated (HCN) channels in the rat ovary are dependent on the type of cell and the reproductive age of the animal: a laboratory investigation. Reprod Biol Endocrinol, 2008, 6:35
36. Dibattista M, Mazzatenta A, Grassi F, et al. Hyperpolarization-activated cyclic nucleotide-gated channels in mouse vomeronasal sensory neurons. J Neurophysiol, 2008, 100(2): 576-586
37. Bolivar JJ, Tapia D, Arenas G, et al. A hyperpolarization-activated, cyclic nucleotide-gated, (Ih-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells. Am J Physiol Cell Physiol, 2008, 294(4): C893-906
38. Ludwig A, Zong X, Jeglitsch M, et al. A family of hyperpolarization-activated mammalian cation channels. Nature, 1998, 393(6685): 5872591
39. Robinson RB, Siegelbaum SA. Hyperpolarization-activated cation currents: from molecules to physiological function. Ann RevPhysiol, 2003, 65:453-480
40. Accili EA, Proenza C, Baruscotti M, et al. From fuuny current to HCN channels: 20 years of excitation. News Physiol. Sci, 2002, 17:32-37
41. Liu J, Dobrzynski H, Yanni J, et al. Organisation of the mouse sinoatrial node: structure and expression of HCN channels. Cardiovasc Res, 2007, 73(4): 729-738
42. Cheng L, Kinard K, Rajamani R, et al. Molecular Mapping of the Binding Site for a Blocker of Hyperpolarization-Activated, Cyclic Nucleotide-Modulated Pacemaker Channels. J. Pharmacol. Exp. Ther, 2007, 322(3): 931-939
43. Whitaker GM, Angoli D, Nazzari H, et al. HCN2 and HCN4 Isoforms Self-assemble and Co-assemble with Equal Preference to Form Functional Pacemaker Channels. J. Biol. Chem., 2007, 282(31): 22900 - 22909
44. Xue T, Siu CW, Lieu DK, et al. Mechanistic Role of If Revealed by Induction of Ventricular Automaticity by Somatic Gene Transfer of Gating-Engineered Pacemaker (HCN) Channels. Circulation, 2007, 115(14): 1839-1850
45. Shan C, Jing W, Steven AS, et al. Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCNl and HCN2 subunits and basalmuodulation by cyclic nucleotide. J Gen Physiol, 2001, 117(5): 491-503
46. Carlo V, Claudia A, Annalisa B, et al. C terminus-mediated control of voltage and cAMP gating of hyperpolarization-activated cyclic nucleotide-gated channels. J Biol Chem, 2001, 276(32): 29930-29934
47. Chen J, Piper DR, Sanguinetti MC, et al. Voltage sensing and activation gating of HCN pacemaker channels. Trends Cardiovasc Med, 2002, 419(6909): 837-841
48. Altomare C, Terragni B, Brioschi C. Heteromeric HCN1-HCN4 channels: a comparison with native pacemaker channels from the rabbit sinoatrial node. J Physiol, 2003,549(Pt2): 347-359
49. Zhang H, Dobrzynskl H, Kodama I, et al. How does the sinoatrial node drive the atrium? J Physiol, 2001, 536:69
50. Matsuura H, Ehara T, Fing WG, et al. Rapidly and slowly activating component of delayed rectifier K+ current in guinea-pig sinoatrial node pacemaker cells. J Physiol,2002, 540(3): 815-820
51. Kyoichi O, Shigehiro S, Toshihiko I, et al. Properties of the delayed rectifier potassium sinoatrial node cells. J Physiol, 2000, 524(1): 51-60
52. Stieber J, Herrmann S, Feil S, et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci USA, 2003,100(25):15235-15240
53. Schulze-Bahr E, Neu A, Friederich P, et al. Pacemaker channel dysfunction in a patient with sinus node disease. J Clin Invest, 2003, 111(10): 1537-1545
54. Er F, Larbig R, Ludwig A, et al. Dominant-negative suppression of HCN channels markedly reduces the native pacemaker current If and undermines spontaneous baeting of neonatal cardiomyocytes. Circulation, 2003, 107:485-489
55. Ueda K, Nakamura K, Hayashi T, et al. Functional characerization of trafficking-defective HCN4 mutation, D553N, associated with cardiac arrhythmia. J Biol Chem, 2004, 279(26): 27194-27198
56. Milanesi R, Barruscoti M, DiFrancesco D, et al. Familial sinus bradycardia associated with a mutation in the cardiac pacemaker channel. N Engl Med, 2006, 353(2): 151-157
57. Zhang Z, Xu YF, Song HT, et al. Functional roles of Cav1.3(a, D) Calcium channel in sinoatrial nodes-insight gained using gene-targeted null mutant mice. Circ Res, 2002, 90:981-987
58. Koschak A, Reimer D, Huber L, et al. alD(Cav 1.3) subunits can from L-type Ca2+ channels activating at negative voltages. J Biol Chem, 2001,276:22100-22106
59. Honjo H, Inada S, Lancaster MK, et al. Sarcoplasmic reticulum Ca2+ release is not a dominating factor in sinoatrial node pacemaker activity at the diastolic potential range in rabbit sinoatrial node cells. Circ Res, 2003, 92:e41-e44
60. Martin Biel, Angela Schneider, Christian Wahl. Cardiac HCN channels, structure, function, and mudulation. Trends in cardiovascular medicine, 2002, 12(5): 206
61. Ruhparwar A, Tebbenjohanns J, Niehaus M, et al. Transplanted fetal cardiomyocytes as cardiac pacemaker. Criculation, 2001, 104(17): Ⅱ335
62. Ruhparwar A, Niehaus M, Radke K, et al. Enrichment of cells of the cardiac conduction system: Neuregulinl enhance the expression of connexin 40 in embryonic cardiomyocytes. Circulation, 2003, 108(17): Ⅳ240
63. Xiao Yong-Fu, Sigg Daniel C. Biological approaches to generating cardiac biopacemaker for bradycardia. Acta Physiologica Sinica, 2007, 59 (5): 562-570
64. Ohnishi S, Nagaya N. Prepare cells to repair the heart: mesenchymal stem cells for the treatment of heart failure. Am J Nephrol, 2007, 27(3): 301-307
65. Niemeyer P, Krause U, Kasten P, et al. Mesenchymal stem cell-based HLA-independent cell therapy for tissue engineering of bone and cartilage. Curr Stem Cell Res Ther, 2006,1(1): 21-27
66. Guo L, Kawazoe N, Fan Y, et al. Chondrogenic differentiation of human mesenchymal stem cells on photoreactive polymer-modified surfaces. Biomaterials, 2008, 29(1):23-32
67. DiFrancesco D, Borer JS. The funny current: cellular basis for the control of heart rate.Drugs, 2007, 67(2): 15-24
68. Kim HT, Kim IS, Lim SE, et al. Gene and cell replacement via neural stem cells. Yonsei Med J,2004, 45: 32-40
69. Fogle KJ, Lyashchenko AK, Turbendian HK, et al. HCN pacemaker channel activation is controlled by acidic lipids downstream of diacylglycerol kinase and phospholipase A2. JNeurosci, 2007, 27(11): 2802-2814.
70. Phillip Pian, Annalisa Bucchi, Richard B. Robinson, et al. Regulation of Gating and Rundown of HCN Hyperpolarization-activated Channels by Exogenous and Endogenous PIP2. The Rockefeller University Press, 2006, 128(5): 593-604
71. Lieu Deborah K, Chan Yau Chi, Lau Chu Pak, et al.Overexpression of HCN-encoded pacemaker current silences bioartificial pacemakers. Heart rhythm : the official journal of the Heart Rhythm Society, 2008, 5(9): 1310-1317
72. Akhavan A. Contribution of pacemaker channels to autonomous electrical activity of differentiated embryonic stem cells. J. Physiol, 2008, 586(10): 2425 - 2426
73. Bolivar JJ, Tapia D, Arenas G, et al. A hyperpolarization-activated, cyclic nucleotide-gated, (Ih-like) cationic current and HCN gene expression in renal inner medullary collecting duct cells. Am J Physiol Cell Physiol, 2008, 294(4): C893 - C906
74. Qu Y, Whitakcr GM, L. Hove-Madsen, et al. Hyperpolarization-activated cyclic nucleotide-modulated 'HCN' channels confer regular and faster rhythmicity to beating mouse embryonic stem cells. J. Physiol., 2008, 586(3): 701 - 716
75. Baruscotti M, Robinson R. B. Electrophysiology and pacemaker function of the developing sinoatrial node. Am J Physiol Heart Circ Physiol, 2007, 293(5): H2613-H2623
76. El Chemaly A, Magaud C, Patri S, et al. The heart rate-lowering agent ivabradine inhibits the pacemaker current I(f) in human atrial myocytes. J Cardiovasc Electrophysiol, 2007, 18(11): 1190-1196
77. Siu CW, Lieu DK, Li RA. HCN-encoded pacemaker channels: from physiology and biophysics to bioengineering. J Membr Biol, 2006, 214(3): 115-122
78. Xue T, Siu CW, Lieu DK, et al. Mechanistic role of I(f) revealed by induction of ventricular automaticity by somatic gene transfer of gating-engineered pacemaker (HCN) channels. Circulation, 2007, 115(14): 1839-50
79. Zhou YF, Yang XJ, Li HX. Hyperpolarization-activated cyclic nucleotide-gated channel gene: the most possible therapeutic applications in the field of cardiac biological pacemakers. Med Hypotheses, 2007, 69(3):541-544
80. Yerra L, Reddy PC. Effects of electromagnetic interference on implanted cardiac devices and their management. Cardiol Rev, 2007, 15(6): 304-309
81. Eaton MJ. Cell and molecular approaches to the attenuation of pain after spinal cord injury. J Neurotrauma, 2006, 23(3-4):549-559
82. Mangoni ME, Nargeot J. Genesis and Regulation of the Heart Automaticity. Physiol Rev, 2008, 88(3): 919-982
1. Edelberg JM, Aird WC, Rosenberg RD. Enhancement of murine cardiac chronotropy by the molecular transfer of the human beta2 adrenergic receptor cDNA. J Clin Invest, 1998, 101(2): 337-343
2. Glenn CM, Pogwizd SM. Gene therapy to develop a genetically engineered cardiac pacemaker. J Cardiovasc Nurs, 2003, 18(5): 330-336
3. Qu J, Plotnikov AN, Danilo P Jr, et al. Expression and function of a biological pacemaker in canine heart. Circulation, 2003, 7(8): 1106-1109
4. Plotnikov AN, Sosunov EA, Qu J, et al. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation, 2004, 109(4): 506-512
5. Mujtaba T, Piper DR, Kalyani A, et al. Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells. Dev Biol, 1999, 214(1):113-127
6. Keller G, Wall C, Fong AZ, et al. Overexpression of H0X11 leads to the immortalization of embryonic precursors with both primitive and definitive hematopoietic potential. Blood, 1998, 92(3): 877-887
7. Noboru Sato, Laurent Meijer, Leandros Skaltsounis, et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nature medicine, 2004, 10:55-63
8. Lisa M Hoffman, Melissa K Carpenter, et al. Characterization and culture of human embryonic stem cells. Nature biotechnology, 2005, 23:699-708
9. Yasushi Takagi, Jun Takahashi, Hidemoto Saiki, et al. Dopaminergic neurons generated from monkey embryonic stem cells function in a parkinson primate model. J. Clin.Invest, 2005, 115(1): 102-109
10. Przemyslaw Blyszczuk, Jaroslaw Czyz, Gabriela Kania, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. PNAS, 2003, 100(3): 998-1003
11. Thomas P. Zwaka, James A. Thomson. Homologous recombination in human embryonic stem cells. Nature biotechnology, 2003, 21: 319-321
12. Chad A. Cowan, Irina Klimanskaya, Jill McMahon, et al. Derivation of Embryonic Stem-Cell Lines from Human Blastocysts. The new England Journal of medine, 2004,350(13): 1353-1356
13. Lisa M Hoffman & Melissa K Carpenter.Characterization and culture of human embryonic stem cells. Nrture Biotechnology, 2005, 23:699-708
14. Assady S, Maor G, Amit M, et al. Insulin production by human embryonic stem cells. Diabetes, 2001, 50(8): 1691-1697
15. Fijnvandraat AC, De Boer PA, Deprez RH, et al. Non- radioactive in situ detection of mRNA in ES cell-derived cardiomyocytes and in the developing heart. Microsc Res Tech, 2002, 58(5): 387-394
16. Kawasaki H, Suemori H, Mizuseki K, et al. Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci U S A,2002, 9(3): 1580-1585
17. Ruhparwar A, Tebbenjohanns J, Niehaus M, et al. Transplanted fetal cardiomyocytes as cardiac pacemaker. Eur J Cardiothorac Surg, 2002, (5): 53-57
18. Ruhparwar A, Haverich A. Prospects for iological cardiac pacemaker systems. Pacing Clin Electrophysiol, 2003, (11): 2069-207
19. Kehat I, Khimovich L, Caspi O, et al. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat Biotechnol, 2004 (10): 1282-1289
20. Tian Xue, Hee Cheol Cho, Fadi G. Akar, et al. Functional Integration of Electrically Active Cardiac Derivatives From Genetically Engineered Human Embryonic Stem Cells With Quiescent Recipient Ventricular. Circulation, 2005, 112(6): e82- e83
21. Ohnishi S, Nagaya N. Prepare cells to repair the heart: mesenchymal stem cells for the treatment of heart failure. Am JNephrol, 2007, 27(3): 301-307
22. Niemeyer P, Krause U, Kasten P, et al. Mesenchymal stem cell-based HLA-independent cell therapy for tissue engineering of bone and cartilage. Curr Stem Cell Res Ther,2006, 1(1): 21-27
23. Guo L, Kawazoe N, Fan Y, et al. Chondrogenic differentiation of human mesenchymal stem cells on photoreactive polymer-modified surfaces. Biomaterials, 2008, 29(1):23-32
24. Valiunas V, Doronin S, Valiuniene L, et al. Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. J Physiol, 2004, (55): 617-626
25. Potapova I, Plotnikov A, Lu Z, et al. Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers. Circ Res, 2004, 4(7): 952-959
26. Beeres SL, Atsma DE, van der Laarse A,et al.Human adult bone marrow mesenchymal stem cells repair experimental conduction block in rat cardiomyocyte cultures. J Am Coll Cardiol, 2005, 6(10): 1943-1952
27. Alexei N. Plotnikov, Eugene A. Sosunov, et al. Biological Pacemaker Implanted in Canine Left Bundle Branch Provides Ventricular Escape Rhythms That Have Physiologically Acceptable Rates. Circulation, 2004, 5(9): 506-512
28. Plotnikov AN, Sosunov EA, Qu J, et al. Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates. Circulation, 2004, (4): 506-512
29. Stieber J, Hofmann F, Ludwig A. Pacemaker channels and sinus node arrhythmia.Trends Cardiovasc Med, 2004, (41): 23-28
30. Ueda K, Nakamura K, Hayashi T, et al. Functional charac- terization of a trafficking-defective HCN4 mutation, D553N, associatedu with cardiac arrhythmia. J Biol Chem, 2004, 79(26): 27194-27198
31. Stieber J, Herrmann S, Feil S, et al. The hyperpolarization- activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci U S A, 2003, 100(25): 15235-15240.
32. Garcia-Frigola C, Shi Y, et al. Expression of the hyperpo- larization-activated cyclic nucleotide-gated cation channel HCN4 during mouse heart development. Gene Expr Patterns, 2003, (6): 777-783
33. Zicha S, Fernandez-Velasco M, Lonardo G, et al. Sinus node dysfunction and hyperpolarization-activated (HCN) channel subunit remodeling in a canine heart failure model. Cardiovasc Res, 2005, 6(3): 430-432
34. Mark F. Pittenger, Alastair M. Mackay, Stephen C. Beck, et al. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science, 1999, 284(5411): 143-147
35. James A. Thomson, Joseph Itskovitz-Eldor, Sander S., et al. Embryonic Stem Cell Lines Derived from Human Blastocysts. Science, 1998, 282(5391): 1145 - 1147
36. Eric Lagasse, Heather Connors. Muhsen Al-Dhalimy, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo .Nature Medicine, 2000, 6: 1229 - 1234
37. Fred H. Gage. Mammalian Neural Stem Cells. Science, 2000, 287(5457): 1433-1438
38. Imhof Alexander, Balajee S. Arunmozhi, Fredricks David N., et al. Breakthrough Fungal Infections in Stem Cell Transplant Recipients Receiving Voriconazole. Clinical Infectious Diseases, 2004, 39: 743-746
39. Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood, 1993, 81(11): 2844-2853
40. Luciano C. Amado, Anastasios P. Saliaris, Karl H. Schuleri, et al. Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. PNAS, 2005, 102(32): 11474-11479
41. Andreas Sch(a|¨)ffler, Christa B(u|¨)chler, et al.Concise Review: Adipose Tissue-Derived Stromal Cells - Basic and Clinical Implications for Novel Cell-Based Therapies. Stem cells, 2007, 25(4): 818-827
42. Massimiliano Gnecchi, Huamei He, Olin D Liang, et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells. Nature medine,2005, 11:367-368
43. Lakshmipathy U, Verfaillie C. Stem cell plasticity. Blood Reviews, 19(1) :29-38
44. Catalin Toma, Mark F. Pittenger, Kevin S. Cahill, et al. Human Mesenchymal Stem Cells Differentiate to a Cardiomyocyte Phenotype in the Adult Murine Heart.Circulation, 2002, 105:93
45. Mark F. Pittenger, Alastair M. Mackay, Stephen C. Beck, et al. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science,1999, 284(5411):143-147
46. Siu CW, Lieu DK, Li RA. HCN-encoded pacemaker channels: from physiology and biophysics to bioengineering. J Membr Biol, 2006, 214(3): 115-122
47. Xue T, Siu CW, Lieu DK, et al. Mechanistic role of I(f) revealed by induction of ventricular automaticity by somatic gene transfer of gating-engineered pacemaker (HCN) channels. Circulation, 2007, 115(14): 1839-50
48. Zhou YF, Yang XJ, Li HX. Hyperpolarization-activated cyclic nucleotide-gated channel gene: the most possible therapeutic applications in the field of cardiac biological pacemakers. Med Hypotheses, 2007, 69(3): 541-544
49. Yerra L, Reddy PC. Effects of electromagnetic interference on implanted cardiac devices and their management. Cardiol Rev, 2007, 15(6): 304-309
50. Verkerk AO, Wilders R, van Borren MM, et al.Pacemaker current I(f) in the human sinoatrial node. Eur Heart J, 2007, 28(20):2472-2478
51. DiFrancesco D, Borer JS. The funny current: cellular basis for the control of heart rate.Drugs, 2007, 67(2): 15-24
52. Xiao Yong-Fu, Daniel C. Sigg. Biological approaches to generating cardiac biopacemaker for bradycardia . Acta Physiologica Sinica, 2007, 59 (5): 562-570