体外诱导小鼠骨髓CD45-细胞向窦房结起搏样细胞定向分化
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摘要
目的探讨体外诱导小鼠骨髓CD45-细胞向窦房结起搏样细胞定向分化的方法。方法免疫磁珠分选小鼠骨髓CD45-细胞,体外培养扩增后,采用添加了5-氮胞苷和小鼠窦房结组织培养上清液的诱导培养基培养,经免疫荧光染色检测起搏样细胞标志物神经丝蛋白-160(NF-160)和超级化激活环核苷酸门控阳离子通道(HCN)4表达,并采用q RT-PCR定量检测起搏基因NF-160、HCN4和HCN2表达。结果小鼠骨髓CD45-细胞经体外诱导培养后得到的分化细胞表达NF-160和HCN4;q RT-PCR分析显示,与体外培养的小鼠窦房结细胞比较,分化起搏样细胞NF-160和HCN4的表达升高,HCN2的表达降低。结论体外培养能够将小鼠骨髓CD45-细胞诱导分化为窦房结起搏样细胞,为构建生物起搏器种子细胞提供选择。
Objective To study method on inducing directional differentiation of mouse bone marrow CD45-cells into sinoatrial node pacemaker-like cells in vitro. Methods Mouse bone marrow CD45-cells were derived by using immunomagnetic beads system. After amplification in vitro, induced medium supplemented with 5-azacytidine and culture supernatant liquid of mouse sinoatrial node cells was used to cultivate. The expression on pacemaker-like cells markers of neurofilament protein(NF-160) and hyperpolarization activated cyclic nucleotide-gated potassium channel(HCN) 4were detected by immunofluorescence staining. The expression of pacing genes(NF-160, HCN4 and HCN2) were detected by q RT-PCR. Results Differentiated cells of bone marrow CD45-cells after induced by culture in vitro expressed NF-160 and HCN4. q RT-PCR analysis showed, compared with cultured sinoatrial node cells in vitro, the expression of NF-160 and HCN4 were higher, while expression of HCN2 was lower. Conclusion The sinoatrial node pacemaker-like cells can be induced and differentiated from mouse bone marrow CD45-cells, and the cells will be one choice of ideal seeding cells for biological pacemaker building.
Objective To study method on inducing directional differentiation of mouse bone marrow CD45-cells into sinoatrial node pacemaker-like cells in vitro. Methods Mouse bone marrow CD45-cells were derived by using immunomagnetic beads system. After amplification in vitro, induced medium supplemented with 5-azacytidine and culture supernatant liquid of mouse sinoatrial node cells was used to cultivate. The expression on pacemaker-like cells markers of neurofilament protein(NF-160) and hyperpolarization activated cyclic nucleotide-gated potassium channel(HCN) 4were detected by immunofluorescence staining. The expression of pacing genes(NF-160, HCN4 and HCN2) were detected by q RT-PCR. Results Differentiated cells of bone marrow CD45-cells after induced by culture in vitro expressed NF-160 and HCN4. q RT-PCR analysis showed, compared with cultured sinoatrial node cells in vitro, the expression of NF-160 and HCN4 were higher, while expression of HCN2 was lower. Conclusion The sinoatrial node pacemaker-like cells can be induced and differentiated from mouse bone marrow CD45-cells, and the cells will be one choice of ideal seeding cells for biological pacemaker building.
引文
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[2]Sah R,Mesirca P,Mason X,et al.Timing of myocardial trpm7 deletion during cardiogenesis variably disrupts adult ventricular function,conduction,and repolarization[J].Circulation,2013,128(2):101-114.
[3]马立军,张勤.急诊心律失常中应用体外无创性心脏起搏的作用综合评价[J].中国现代医生,2014,52(9):31-33.
[4]Aanhaanen WT,Mommersteeg MT,Norden J,et al.Developmental origin,growth,and three-dimensional architecture of the atrioventricular conduction axis of the mouse heart[J].Circ Res,2010,107(6):728-736.
[5]Hashem SI,Claycomb WC.Genetic isolation of stem cellderived pacemaker-nodal cardiac myocytes[J].Mol Cell Biochem,2013,383(1-2):161-171.
[6]Ye SX,Qu Y,Dan P,et al.Isolation and characterization of atrioventricular nodal cells from neonate rabbit heart[J].Circ Arrhythm Electrophysiol,2011,4(6):936-946.
[7]Vincent SD,Buckingham ME.How to make a heart:the origin and regulation of cardiac progenitor cells[J].Curr Top Dev Biol,2010,90:1-41.
[8]Makino S,Fukuda K,Miyoshi S,et al.Cardiomyocytes can be generated from marrow stromal cells in vitro[J].J Clin Invest,1999,103(5):697-705.
[9]Pauza DH,Saburkina I,Rysevaite K,et al.Neuroanatomy of the murine cardiac conduction system:a combined stereomicroscopic and fluorescence immunohistochemical study[J].Auton Neurosci,2013,176(1-2):32-47.
[10]Ou Y,Niu XL,Ren FX.Expression of key ion channels in the rat cardiac conduction system by laser capture microdissection and quantitative real-time PCR[J].Exp Physiol,2010,95(9):938-945.
[11]Greener ID,Monfredi O,Inada S,et al.Molecular architecture of the human specialised atrioventricular conduction axis[J].J Mol Cell Cardiol,2011,50(4):642-651.
[12]Pauza DH,Rysevaite K,Inokaitis H,et al.Innervation of sinoatrial nodal cardiomyocytes in mouse.a combined approach using immunofluorescent andelectron microscopy[J].J Mol Cell Cardiol,2014,75:188-197.
[13]Moretti A,Caron L,Nakano A,et al.Multipotent embryonic isl1+progenitor cells lead to cardiac,smooth muscle,and endothelial cell diversification[J].Cell,2006,127(6):1151-1165.
[14]Spter D,Abramczuk MK,Buac K,et al.A HCN4+cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells[J].Nat Cell Biol,2013,15(9):1098-1106.
[15]Yamamoto M,Dobrzynski H,Tellez J,et al.Extended atrial conduction system characterized by the expression of the HCN4 channel and connexin45[J].Cardiovasc Res,2006,72(2):271-281.
[16]Garcia-Frigola C,Shi Y,Evans SM.Expression of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 during mouse heartdevelopment[J].Gene Expr Patterns,2003,3(6):777-783.
[17]Herrmann S,Layh B,Ludwig A.Novel insights into the distribution of cardiac HCN channels:an expression study in the mouse heart[J].J Mol Cell Cardiol,2011,51(6):997-1006.