改良的乳鼠原代心房肌细胞的培养及鉴定方法
|
摘要
目的:主要探讨原代心房肌细胞的培养及鉴定方法,为进一步研究心房颤动的重构机制及治疗方法奠定基础。方法:选取1-3 d的SD乳鼠40只,雌雄不限,分离心房、心室肌,胰酶联合EDTA充分消化心房肌细胞,利用心房肌与成纤维细胞的差速贴壁及细胞传代方法纯化心房肌细胞,免疫细胞化学染色鉴定心房肌细胞。结果:心房肌细胞培养至第3天,可见心房肌细胞覆盖率高达90%,并出现波动性,免疫细胞化学染色可见90%的心房肌细胞肌经α-肌动蛋白抗体染色阳性。结论:经酶化学消化法可成功培养出原代心房肌细胞,是一种较好的培养及鉴定乳鼠心房肌细胞的方法。
Objective: To investigate the culture and identification of major primary atrial myocytes, which can be used to study the remodelling of atrium or relative future research. Methods: Atrial and ventricular muscle is isolated from 40 Sprague Dawley rats(1-3days old, male or female), trypsin EDTA is added to digest fully, then atrial myocytes were purified with differential adhesion method and passage and identified with immunocytochemistry. Results: Atrial myocytes coverage are up to 90% after 72 hours primary culture, and show the feature of volatility, 90% of myocytes of atrium can be authenticated by α-actin antibody staining with immunocytochemistry.Conclusion: Atrial myocytes with high purity and vigor can be separated by enzyme digestion, it is a better method of cultivation and identification of atrial myocytes of neonatal rat.
Objective: To investigate the culture and identification of major primary atrial myocytes, which can be used to study the remodelling of atrium or relative future research. Methods: Atrial and ventricular muscle is isolated from 40 Sprague Dawley rats(1-3days old, male or female), trypsin EDTA is added to digest fully, then atrial myocytes were purified with differential adhesion method and passage and identified with immunocytochemistry. Results: Atrial myocytes coverage are up to 90% after 72 hours primary culture, and show the feature of volatility, 90% of myocytes of atrium can be authenticated by α-actin antibody staining with immunocytochemistry.Conclusion: Atrial myocytes with high purity and vigor can be separated by enzyme digestion, it is a better method of cultivation and identification of atrial myocytes of neonatal rat.
引文
[1]Heeringa J,van der Kuip DA,Hofman A,et al.Prevalence,incidence and lifetime risk of atrial fibrillation:the Rotterdam study[J].Eur Heart,2006,27(8):949-953
[2]Wakili R,Voigt N,Kaab S,et al.Recent advances in the molecular pathophysiology of atrial fibrillation[J].Clin Invest,2011,121(8):2955-2968
[3]Nattel S,Burstein B,Dobrev D.Atrial remodeling and atrialfibrillation:mechanisms and implications[J].Circ Arrhythm Electrophysiol,2008,1:62-73
[4]Nattel S,Dobrev D.The multidimensional role of calcium in atrial fibrillation pathophysiology:mechanistic insights and therapeutic Opportunities[J].Eur Heart,2012,33:1870-1877
[5]Nattel S,Harada M.Atrial remodeling and atrial fibrillation:recent advances and translational perspectives[J].Am Coll Cardiol,2014,63(22):2335-2345
[6]Bird SD,Doevendans PA,Rooijen MA,et al.The human adult cardiomyocyte phenotype[J].Cardiovasc Res,2003,8(2):423
[7]Marshall GE,Russell JA,Tellez JO,et al.Remodelling of human atrial K+currents but not ino channel expreeion by chronicβ-blockade[J].Pfuger Arch,2012,463(4):537-548
[8]Saygili E,Rana OR,Saygili E,et al.Losartan prevents atretch-induced electrical remodeling in cultured artial neonatal myocytes[J].AM J Physiol Heart Circ Physiol,2007,292(6):H2898-H2905
[9]Saygili E,Rana OR,Meyer C,et al.The angiotensin-calcineurin-NFAT pathway mediates stretch-induced upregulation of matrix metalloproteinases-2/-9 in artial myocytes[J].J Basic Res Cardiol,2009,104(4):435-448
[10]Li Q,O'Nell SC,Tao T,et al.Mechanises ba which cytoplasmic calcium wave propagation and alterans are generated in cardiac artial myocytes lacking T-tubules-insights from a simulation study[J].Biophys J,2012,102(7):1471-1482
[11]Collings TP,Terrar DA.Ca2+-stimulated adenylyl cyclases regulate the L-type Ca2+current in guinea-pig atrial myocytes[J].J Physiol,2012,590(8):1881-1893
[12]Shkryl VM,Maxwell JT,Domeier TL,et al.Refractoriness of sarcoplasmic reticulum Ca2+release determines Ca2+alternans in artial myocytes[J].Am J Physiol Heart Circ Physiol,2012,302(11):2310-2320
[13]Hinton M,Mellow L,Halayko AJ,et al.Hypoxia induces hypersensitivity and hyperreactivity to thromboxane receptor agonist in neonatal pulmonary arterial myocytes[J].Am J Physiol Lung Cell Mol Physiol,2006,290(2):L375-384
[14]刘雄涛,苏菲菲,尚福军,等.人类心房肌细胞的原代培养与鉴定[J].科学技术与工程,2014,2(14):5-7Liu Xiong-tao,Su Fei-fei,Shang Fu-jun,et al.Ptimary culture and identification of artial myocytes of human[J].Science,Technology,Engineering,2014,2(14):5-7
[15]罗淋,刘志琴,杨龙,等.大鼠乳鼠原代心房肌细胞培养的方法及鉴定[J].国际心血管病杂志,2013,2(40):112-115Luo Lin,Liu Zhi-qin,Yang Long,et al.Culture and idengtification of primary artial myocytes in neonatal rat[J].International Journal of cardiovascular disease,2013,2(40):112-115
[16]王志勇,周立,郭义威,等.乳鼠心肌细胞的培养改进[J].医学信息(内、外科版),2009,3(22):240-241Wang Zhi-yong,Zhou Li,Guo Yi-wei,et al.The cultivation of the rats myocardial cells[J].Medical information(internal and surgical version),2009,3(22):240-241
[17]Miragoli M,Gaudesius G,Rohr S.Electrotonic modulation of cardiac impulse conduction by myofibroblasts[J].Circ Res,2006,98(6):801-810
[18]Hsua KL,Fanb H,Chen YC,et al.Protein kinase C-Fyn kinase cascade mediates the oleic acid-induced disassembly of neonatal rat cardiomyocyte adherens junctions[J].Int J Biochem Cell Bio,2009,41(7):1536-1546
[19]王伟,肖颖彬,程伟.快速电场起搏对心房肌细胞电生理特性的实验研究[J].重庆医学,2010,39(14):1792-1797Wang Wei,Xiao Ying-bin,Chen Wei.Study of rapid pacing-Induced electrical remodeling in cultured neonatal rat atrial myocytes[J].Chongqing Medical,2010,39(14):1792-1797
[20]乐智勇,罗红.乳鼠心肌细胞体外培养的探讨[J].湖北中医学院学报,2007,9(3):33-34Yue Zhi-yong,Luo Hong.Culture in vitro of neonatal rat cardiac meyocytes[J].Journal of Hubei college of TCM,2007,9(3):33-34
[21]郭勇,张西正,魏严,等.新生大鼠心肌细胞的体外培养和生物学观察[J].生物医学工程研,2006,25(4):259-262Guo Yong,Zhang Xi-zheng,Wei Yan,et al.Culture in vitro and observation of neonatal rat cardiac myocytes[J].Journal of biomedical engineeringresearch,2006,25(4):259-262
[22]郎明健,郭敏,曾秋棠,等.差速贴壁联合应用5-Brd U对心肌细胞纯度及活力的影响[J].心脏杂志,2008,20(5):566-569Lang Ming-jian,Guo Min,Zeng Qiu-tang,et.al.Effects of repeatedly differential attachment technique combined with 5-B rd U on the purity and the activity of isolated cardiomyocytes[J].Chin Heart J,2008,20(5):566-569
[23]Sun H,Chartier D,Leblanc N,et al.Intracelluar calcium changes and tachycardia-induced contractile dysfunction in ca nineatrial myoytes[J].Cardiovasc Res,2001,49(4:)751-761
[24]Moeller A1,Ask K,Warburton D,et al.The bleomycin animal model:a useful tool to investigate treatment options for idiopathic pulmonary fibrosis[J].Int J Biochem Cell Biol,2008,40(3):362-382
[2]Wakili R,Voigt N,Kaab S,et al.Recent advances in the molecular pathophysiology of atrial fibrillation[J].Clin Invest,2011,121(8):2955-2968
[3]Nattel S,Burstein B,Dobrev D.Atrial remodeling and atrialfibrillation:mechanisms and implications[J].Circ Arrhythm Electrophysiol,2008,1:62-73
[4]Nattel S,Dobrev D.The multidimensional role of calcium in atrial fibrillation pathophysiology:mechanistic insights and therapeutic Opportunities[J].Eur Heart,2012,33:1870-1877
[5]Nattel S,Harada M.Atrial remodeling and atrial fibrillation:recent advances and translational perspectives[J].Am Coll Cardiol,2014,63(22):2335-2345
[6]Bird SD,Doevendans PA,Rooijen MA,et al.The human adult cardiomyocyte phenotype[J].Cardiovasc Res,2003,8(2):423
[7]Marshall GE,Russell JA,Tellez JO,et al.Remodelling of human atrial K+currents but not ino channel expreeion by chronicβ-blockade[J].Pfuger Arch,2012,463(4):537-548
[8]Saygili E,Rana OR,Saygili E,et al.Losartan prevents atretch-induced electrical remodeling in cultured artial neonatal myocytes[J].AM J Physiol Heart Circ Physiol,2007,292(6):H2898-H2905
[9]Saygili E,Rana OR,Meyer C,et al.The angiotensin-calcineurin-NFAT pathway mediates stretch-induced upregulation of matrix metalloproteinases-2/-9 in artial myocytes[J].J Basic Res Cardiol,2009,104(4):435-448
[10]Li Q,O'Nell SC,Tao T,et al.Mechanises ba which cytoplasmic calcium wave propagation and alterans are generated in cardiac artial myocytes lacking T-tubules-insights from a simulation study[J].Biophys J,2012,102(7):1471-1482
[11]Collings TP,Terrar DA.Ca2+-stimulated adenylyl cyclases regulate the L-type Ca2+current in guinea-pig atrial myocytes[J].J Physiol,2012,590(8):1881-1893
[12]Shkryl VM,Maxwell JT,Domeier TL,et al.Refractoriness of sarcoplasmic reticulum Ca2+release determines Ca2+alternans in artial myocytes[J].Am J Physiol Heart Circ Physiol,2012,302(11):2310-2320
[13]Hinton M,Mellow L,Halayko AJ,et al.Hypoxia induces hypersensitivity and hyperreactivity to thromboxane receptor agonist in neonatal pulmonary arterial myocytes[J].Am J Physiol Lung Cell Mol Physiol,2006,290(2):L375-384
[14]刘雄涛,苏菲菲,尚福军,等.人类心房肌细胞的原代培养与鉴定[J].科学技术与工程,2014,2(14):5-7Liu Xiong-tao,Su Fei-fei,Shang Fu-jun,et al.Ptimary culture and identification of artial myocytes of human[J].Science,Technology,Engineering,2014,2(14):5-7
[15]罗淋,刘志琴,杨龙,等.大鼠乳鼠原代心房肌细胞培养的方法及鉴定[J].国际心血管病杂志,2013,2(40):112-115Luo Lin,Liu Zhi-qin,Yang Long,et al.Culture and idengtification of primary artial myocytes in neonatal rat[J].International Journal of cardiovascular disease,2013,2(40):112-115
[16]王志勇,周立,郭义威,等.乳鼠心肌细胞的培养改进[J].医学信息(内、外科版),2009,3(22):240-241Wang Zhi-yong,Zhou Li,Guo Yi-wei,et al.The cultivation of the rats myocardial cells[J].Medical information(internal and surgical version),2009,3(22):240-241
[17]Miragoli M,Gaudesius G,Rohr S.Electrotonic modulation of cardiac impulse conduction by myofibroblasts[J].Circ Res,2006,98(6):801-810
[18]Hsua KL,Fanb H,Chen YC,et al.Protein kinase C-Fyn kinase cascade mediates the oleic acid-induced disassembly of neonatal rat cardiomyocyte adherens junctions[J].Int J Biochem Cell Bio,2009,41(7):1536-1546
[19]王伟,肖颖彬,程伟.快速电场起搏对心房肌细胞电生理特性的实验研究[J].重庆医学,2010,39(14):1792-1797Wang Wei,Xiao Ying-bin,Chen Wei.Study of rapid pacing-Induced electrical remodeling in cultured neonatal rat atrial myocytes[J].Chongqing Medical,2010,39(14):1792-1797
[20]乐智勇,罗红.乳鼠心肌细胞体外培养的探讨[J].湖北中医学院学报,2007,9(3):33-34Yue Zhi-yong,Luo Hong.Culture in vitro of neonatal rat cardiac meyocytes[J].Journal of Hubei college of TCM,2007,9(3):33-34
[21]郭勇,张西正,魏严,等.新生大鼠心肌细胞的体外培养和生物学观察[J].生物医学工程研,2006,25(4):259-262Guo Yong,Zhang Xi-zheng,Wei Yan,et al.Culture in vitro and observation of neonatal rat cardiac myocytes[J].Journal of biomedical engineeringresearch,2006,25(4):259-262
[22]郎明健,郭敏,曾秋棠,等.差速贴壁联合应用5-Brd U对心肌细胞纯度及活力的影响[J].心脏杂志,2008,20(5):566-569Lang Ming-jian,Guo Min,Zeng Qiu-tang,et.al.Effects of repeatedly differential attachment technique combined with 5-B rd U on the purity and the activity of isolated cardiomyocytes[J].Chin Heart J,2008,20(5):566-569
[23]Sun H,Chartier D,Leblanc N,et al.Intracelluar calcium changes and tachycardia-induced contractile dysfunction in ca nineatrial myoytes[J].Cardiovasc Res,2001,49(4:)751-761
[24]Moeller A1,Ask K,Warburton D,et al.The bleomycin animal model:a useful tool to investigate treatment options for idiopathic pulmonary fibrosis[J].Int J Biochem Cell Biol,2008,40(3):362-382