骨髓间充质干细胞移植对梗死后大鼠心肌钾离子通道Ito Kv4.2影响的实验研究
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摘要
背景:骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)是一种具有多向分化潜能和自我复制功能的未分化细胞群。大量的实验研究和临床研究均证实移植MSCs能改善梗死后心功能,但移植细胞潜在的致心律失常作用仍是当今心血管领域不容忽视的问题。I_(to)是动作电位发生时第一个复极电流,它包括两个成分:快成分I_(tof)和慢成分I_(tos)。Itof属于Kv4.2或Kv4.3通道电流。有研究表明,在异常条件下,I_(to)是形成2相折返的重要因素1。敲除Kv1.4和Kv4.2的小鼠研究表明,动作电位时程延长,甚至出现了早后去极化(early afterdepolarization,EAD)。
目的:探讨心肌梗死后移植骨髓间充质干细胞对心室肌细胞钾离子通道Ito亚单位Kv4.2基因表达的影响,为MSCs移植治疗心肌梗死和对梗死后心肌电生理影响机制提供理论基础。
方法:采用贴壁培养方法分离和扩增MSCs,使用流式细胞仪检测细胞表面抗原,并鉴定MSCs的成骨、成脂和成心肌分化的多向分化能力。将上述MSCs及其实验对照注射到急性冠状动脉前降支结扎的雄性大鼠左心室前壁,移植后7天观察MSCs在梗死心脏中的定位和存活;14天后程序电刺激(Programmed Electrical Stimulation,PES)测定室性心律失常(Ventricular Arrhythmias,VAs)诱发率;心肌组织HE染色和荧光显微镜观察移植细胞,RT-PCR和Western blot分别检测钾离子通道Ito亚单位Kv4.2基因mRNA和蛋白水平。
结果:1.培养的MSCs表达CD_(44)(94.5%)、CD_(29)(90.7%)、CD_(105)(73.5%),很少表达CD_(34)(3.3%)、CD_(14)(1.2%)、CD_(45)(5.2%)。在合适的培养条件下可以诱导分化成脂肪细胞、骨细胞以及心肌样细胞。2.心肌梗死后两周VAs诱发率增加,MSCs能减少VAs的诱发率,但各组间没有统计学意义。3.心肌组织免疫荧光检测发现,MSCs集中分布于梗死区和梗死心肌周围;4.心肌梗死组和心肌梗死+细胞培养基组Kv4.2 mRNA量和蛋白量明显下降,与假手术组相比有显著差异;心肌梗死+干细胞组Kv4.2mRNA量和蛋白表达量较心肌梗死组明显升高,与心肌梗死组间差异有统计学意义。
结论:MSCs移植减少了VAs诱发率,提高心肌组织中I_(to)亚单位Kv4.2基因表达,改善其分布,可能减少心律失常发生。
Backgrounds: Bone marrow mesenchymal stem cells (MSCs) are undifferentiated multipotent cell population with the potential to be self-renewed. A variety of experiments and clinical studies suggested that MSCs can improve cardiac pump function after acute myocardial infarction. Moreover, concern that intramyocardial transplantation of cells could cause potentially life-threatening ventricular arrhythmias has been repeatedly reported. Transient Outward Potassium Current(Ito) is the first repolarization current when action potential occurs, which includes fast Ito and slow I_(to). The study demonstrated that I_(to) is a important factor of tow phase reentry. The study on rat without genes Kv1.4 and Kv4.2 indicated prolongation of action potential duration, even with early afterdepolarization.
Objectives: To investigate the effects of bone marrow mesenchymal stem cells (MSCs) implantation in postinfarcted rat myocardium on transient outward potassium Kv4.2 of cardiomyocytes in left ventricle, to assess the electrophysiological and arrhythmogenic effects and provide efficacy and safety evidences for MSCs therapy in AMI.
Methods: Male MSCs are cultured and expanded using density gradient centrifugation. Seven days after intracardiac injection into a male rat left anterior descending (LAD) ligation model, cell survival and engraftment were identified by GFP immunofluorescence. Two weeks after transplantation, ventricular arrhythmias (VAs)inducibility were assessed by echocardiography and programmed electrical stimulation(PES). Left ventricular morphology was evaluated through H&E. Transplanted cells were observed by fluorescent microscope. RT-PCR and Western blot were used to identify expression of I_(to) Kv4.2.
Results: 1. MSCs were negative for haemopoietic markers CD_(34),CD_(14) and CD45 and positive for CD_(29), CD_(44), and CD_(105). MSCs cultured in differentiation medium led to Oil red-O-positive or Alizarin Red positive. Part of 5-azacytidine treated MSCs expressed cardiac marker troponin T. 2. Fluorescent microscope showed that MSC-derived cells survived. The expression of Kv4.2 in the MI group and MI-CM group was significantly lower than that in the sham-operating group. Compared to MI-CM group and MI group, expression of Kv4.2 was significantly increased. MSCs injection led to significantly reduced inducibility of VAs.
Conclusion: The MSCs transplantation significantly reduced inducibility of VAs. The MSCs transplantation treatment can elevate the expression of Kv4.2 of AMI rats, which may be related to decrease of arrhythmias after MSCs transplantation.
目的:探讨心肌梗死后移植骨髓间充质干细胞对心室肌细胞钾离子通道Ito亚单位Kv4.2基因表达的影响,为MSCs移植治疗心肌梗死和对梗死后心肌电生理影响机制提供理论基础。
方法:采用贴壁培养方法分离和扩增MSCs,使用流式细胞仪检测细胞表面抗原,并鉴定MSCs的成骨、成脂和成心肌分化的多向分化能力。将上述MSCs及其实验对照注射到急性冠状动脉前降支结扎的雄性大鼠左心室前壁,移植后7天观察MSCs在梗死心脏中的定位和存活;14天后程序电刺激(Programmed Electrical Stimulation,PES)测定室性心律失常(Ventricular Arrhythmias,VAs)诱发率;心肌组织HE染色和荧光显微镜观察移植细胞,RT-PCR和Western blot分别检测钾离子通道Ito亚单位Kv4.2基因mRNA和蛋白水平。
结果:1.培养的MSCs表达CD_(44)(94.5%)、CD_(29)(90.7%)、CD_(105)(73.5%),很少表达CD_(34)(3.3%)、CD_(14)(1.2%)、CD_(45)(5.2%)。在合适的培养条件下可以诱导分化成脂肪细胞、骨细胞以及心肌样细胞。2.心肌梗死后两周VAs诱发率增加,MSCs能减少VAs的诱发率,但各组间没有统计学意义。3.心肌组织免疫荧光检测发现,MSCs集中分布于梗死区和梗死心肌周围;4.心肌梗死组和心肌梗死+细胞培养基组Kv4.2 mRNA量和蛋白量明显下降,与假手术组相比有显著差异;心肌梗死+干细胞组Kv4.2mRNA量和蛋白表达量较心肌梗死组明显升高,与心肌梗死组间差异有统计学意义。
结论:MSCs移植减少了VAs诱发率,提高心肌组织中I_(to)亚单位Kv4.2基因表达,改善其分布,可能减少心律失常发生。
Backgrounds: Bone marrow mesenchymal stem cells (MSCs) are undifferentiated multipotent cell population with the potential to be self-renewed. A variety of experiments and clinical studies suggested that MSCs can improve cardiac pump function after acute myocardial infarction. Moreover, concern that intramyocardial transplantation of cells could cause potentially life-threatening ventricular arrhythmias has been repeatedly reported. Transient Outward Potassium Current(Ito) is the first repolarization current when action potential occurs, which includes fast Ito and slow I_(to). The study demonstrated that I_(to) is a important factor of tow phase reentry. The study on rat without genes Kv1.4 and Kv4.2 indicated prolongation of action potential duration, even with early afterdepolarization.
Objectives: To investigate the effects of bone marrow mesenchymal stem cells (MSCs) implantation in postinfarcted rat myocardium on transient outward potassium Kv4.2 of cardiomyocytes in left ventricle, to assess the electrophysiological and arrhythmogenic effects and provide efficacy and safety evidences for MSCs therapy in AMI.
Methods: Male MSCs are cultured and expanded using density gradient centrifugation. Seven days after intracardiac injection into a male rat left anterior descending (LAD) ligation model, cell survival and engraftment were identified by GFP immunofluorescence. Two weeks after transplantation, ventricular arrhythmias (VAs)inducibility were assessed by echocardiography and programmed electrical stimulation(PES). Left ventricular morphology was evaluated through H&E. Transplanted cells were observed by fluorescent microscope. RT-PCR and Western blot were used to identify expression of I_(to) Kv4.2.
Results: 1. MSCs were negative for haemopoietic markers CD_(34),CD_(14) and CD45 and positive for CD_(29), CD_(44), and CD_(105). MSCs cultured in differentiation medium led to Oil red-O-positive or Alizarin Red positive. Part of 5-azacytidine treated MSCs expressed cardiac marker troponin T. 2. Fluorescent microscope showed that MSC-derived cells survived. The expression of Kv4.2 in the MI group and MI-CM group was significantly lower than that in the sham-operating group. Compared to MI-CM group and MI group, expression of Kv4.2 was significantly increased. MSCs injection led to significantly reduced inducibility of VAs.
Conclusion: The MSCs transplantation significantly reduced inducibility of VAs. The MSCs transplantation treatment can elevate the expression of Kv4.2 of AMI rats, which may be related to decrease of arrhythmias after MSCs transplantation.
引文
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