体外诱导心肌样细胞中HCN4过表达对起搏电流特性的影响
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摘要
目的体外构建具有表达功能性的起搏心肌样细胞,并检测起搏心肌样细胞中起搏电流的特性。方法分离培养大鼠骨髓间充质干细胞诱导分化为心肌样细胞。构建携带人HCN4基因的重组腺病毒载体(rAd/GFP/HCN4)和绿色荧光蛋白的腺病毒载体(rAd/GFP),转染至心肌样细胞分为rAd/GFP/HCN4组和rAd/GFP组,不做任何处理作为对照组。Western blot检测各组HCN4蛋白表达,全细胞膜片钳技术记录各组中起搏电流情况。结果荧光显微镜观察到长梭形心肌样细胞,PCR鉴定重组腺病毒载体rAd/GFP/HCN4和rAd/GFP构建成功;rAd/GFP/HCN4组HCN4蛋白相对表达量(1.31±0.02)高于rAd/GFP组(0.55±0.02)和对照组(0.52±0.03)(P<0.05),rAd/GFP组与对照组比较差异无统计学意义(P>0.05);膜片钳实验在转基因细胞中检测到起搏电流,而rAd/GFP组和对照组中未检测到起搏电流。结论 rAd/GFP/HCN4转染到心肌样细胞中,心肌样细胞中HCN4蛋白表达增加且可检测到起搏电流,从而使心肌样细胞具有自主搏动,为体外构建生物起搏器提供研究基础。
Objective To construct pacing cardiomyocyte-like cells in vitro and to study the characteristics of pacemaker current in cardiomyocyte-like cells.Methods Bone marrow mesenchymal stem cells(BMSCs)were isolated and induced to differentiate into cardiomyocyte-like cells.Cardiomyocyte-like cells transfected by recombinant adenoviral vector(rAd)carrying green fluorescent protein(GFP)and human HCN4 gene were named as rAd/GFP/HCN4 group.Cardiomyocytelike cells transfected by rAd carrying GFP gene were named as rAd/GFP group.Cardiomyocyte-like cells without transfection were as controls(control group).Western blot was used to detect the expression of HCN4 protein in each group,and whole-cell patch clamp technique was used to record the pacemaker current in each group.Results Spindleshaped cardiomyocyte-like cells were observed by fluorescence microscopy.PCR showed that rAd carrying GFP and HCN4 gene were successfully constructed.The relative expression of HCN4 protein in rAd/GFP/HCN4 group(1.31±0.02)was significantly higher than that in rAd/GFP group(0.55±0.02)(P<0.05)and control group(0.52±0.03)(P<0.05),and there was no significant difference between rAd/GFP group and control group(P>0.05).Pacemaker current was found in rAd/GFP/HCN4 group,but was not found in rAd/GFP group and control group by patch clamp technique.Conclusion HCN4 protein is highly expressed and the pacemaker current is found in cardiomyocyte-like cells transfected by rAd/GFP/HCN4,resulting in autonomous pulsation of cardiomyocyte-like cells,which might provide a research basis for creating biological pacemakers in vitro.
Objective To construct pacing cardiomyocyte-like cells in vitro and to study the characteristics of pacemaker current in cardiomyocyte-like cells.Methods Bone marrow mesenchymal stem cells(BMSCs)were isolated and induced to differentiate into cardiomyocyte-like cells.Cardiomyocyte-like cells transfected by recombinant adenoviral vector(rAd)carrying green fluorescent protein(GFP)and human HCN4 gene were named as rAd/GFP/HCN4 group.Cardiomyocytelike cells transfected by rAd carrying GFP gene were named as rAd/GFP group.Cardiomyocyte-like cells without transfection were as controls(control group).Western blot was used to detect the expression of HCN4 protein in each group,and whole-cell patch clamp technique was used to record the pacemaker current in each group.Results Spindleshaped cardiomyocyte-like cells were observed by fluorescence microscopy.PCR showed that rAd carrying GFP and HCN4 gene were successfully constructed.The relative expression of HCN4 protein in rAd/GFP/HCN4 group(1.31±0.02)was significantly higher than that in rAd/GFP group(0.55±0.02)(P<0.05)and control group(0.52±0.03)(P<0.05),and there was no significant difference between rAd/GFP group and control group(P>0.05).Pacemaker current was found in rAd/GFP/HCN4 group,but was not found in rAd/GFP group and control group by patch clamp technique.Conclusion HCN4 protein is highly expressed and the pacemaker current is found in cardiomyocyte-like cells transfected by rAd/GFP/HCN4,resulting in autonomous pulsation of cardiomyocyte-like cells,which might provide a research basis for creating biological pacemakers in vitro.
引文
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[16] NONG Y,ZHANG C,WEI L,et al.In situinvestigation of allografted mouse HCN4 gene transfected rat bone marrow mesenchymal stromal cells with the use of patch-clamp recording of ventricular slices[J].Cytotherapy,2013,15(8):905-919.
[17] YANG X J,ZHOU Y F,LI H X,et al.Mesenchymal stem cells as a gene delivery system to create biological pacemaker cells in vitro[J].J Int Med Res,2008,36(5):1049-1055.
[2] OHKAWA T, FUKATA Y, YAMASAKI M, et al.Autoantibodies to epilepsy-related LGI1in limbic encephalitis neutralize LGI1-ADAM22 interaction and reduce synaptic AMPA receptors[J].J Neurosci,2013,33(46):18161-18174.
[3] IRANI S R,PETTINGILL P,KLEOPA K A,et al.Morvan syndrome:clinical and serological observations in 29cases[J].Ann Neurol,2012,72(2):241-255.
[4] EDELBERG J M, AIRD W C, ROSENBERG R D.Enhancement of murine cardiac chronotropy by the molecular transfer of the human beta2adrenergic receptor cDNA[J].J Clin Invest,1998,101(2):337-343.
[5] PLOTNIKOV A N,SOSUNOV E A,QU J,et al.Biological pacemaker implanted in canine left bundle branch provides ventricular escape rhythms that have physiologically acceptable rates[J].Circulation,2004,109(4):506-512.
[6] HU Y F,DAWKINS J F,CHO H C,et al.Biological pacemaker created by minimally invasive somatic reprogramming in pigs with complete heart block[J].Sci Transl Med,2014,6(245):245-294.
[7] SHI W, WYMORE R, YU H,et al. Distribution and prevalence of hyperpolarization activated cation channel(HCN)mRNA expression in cardiac tissues[J].Circ Res,1999,85(1):e1-6.
[8] BAX N A,VAN MARION M H,SHAH B,et al.Matrix production and remodeling capacity of cardiomyocyte progenitor cells during in vitro differentiation[J].J Mol Cell Cardiol,2012,53(4):497-508.
[9] CHAUVEAU S,BRINK P R,COHEN I S.Stem cell-based biological pacemakers from proof of principle to therapy:a review[J].Cytotherapy,2014,16(7):873-880.
[10]韩博,蒋玉东,董大明.骨髓间充质干细胞治疗脊髓损伤的研究进展[J].中华实用诊断与治疗杂志,2017,31(3):300-302.
[11] JUN C,ZHIHUI Z,LU W,et al.Canine bone marrow mesenchymal stromal cells with lentiviral mHCN4gene transfer create cardiac pacemakers[J].Cytotherapy,2012,14(5):529-539.
[12]张凤香,李树青,孙大鹏,等.5-氮胞苷体外诱导大鼠骨髓基质干细胞向心肌样细胞的分化[J].中国组织工程研究与临床康复,2009,13(49):9701-9704.
[13]马鹰,卫红军,张七一,等.荧光定量RT-PCR法检测超极化激活环核苷酸门控阳离子通道在窦房结中表达[J].中华实用诊断与治疗杂志,2010,24(12):1200-1202.
[14] DOBRZYNSKI H,BOYETT M R,ANDERSON R H.New insights into pacemaker activity:promoting understanding of sick sinus syndrome[J].Circulation,2007,115(14):1921-1932.
[15]温昌霖,张忠良,王国华.孤立性房颤与心脏结构功能相关性研究[J].实用诊断与治疗杂志,2007,21(6):436-437.
[16] NONG Y,ZHANG C,WEI L,et al.In situinvestigation of allografted mouse HCN4 gene transfected rat bone marrow mesenchymal stromal cells with the use of patch-clamp recording of ventricular slices[J].Cytotherapy,2013,15(8):905-919.
[17] YANG X J,ZHOU Y F,LI H X,et al.Mesenchymal stem cells as a gene delivery system to create biological pacemaker cells in vitro[J].J Int Med Res,2008,36(5):1049-1055.