重组慢病毒介导超极化激活的环核苷酸门控阳离子通道4基因转染BMSCs的实验研究_《中国修复重建外科杂志》

作者：

于风旭 , 方易冰 , 刘炫 , 赖前成 , 贾建博 , 廖斌

泸州医学院附属医院心胸外科（四川泸州，646000）;

关键词：

超极化激活的环核苷酸门控阳离子通道4 BMSCs 慢病毒转染起搏电流

DOI：

10.7507/1002-1892.20130331

视频：

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摘要 全文 图表 视频 参考文献 施引文献 补充材料

目的探讨重组慢病毒（lentivirus，LVs）介导超极化激活的环核苷酸门控阳离子通道4（hyperpo-larization-activated cyclic nucleotide-gated cation channel 4，HCN4）基因转染大鼠BMSCs构建生物起搏细胞的可行性。方法选取3～5周龄SD大鼠，采用改良全骨髓贴壁培养法分离培养BMSCs。以LVs作为转染载体，增强绿色荧光蛋白（enhanced green fluorescent protein，EGFP）为标记，构建LVs-HCN4-EGFP病毒液。取第3代BMSCs分别转染LVs-HCN4-EGFP病毒液（实验组）和LVs-EGFP空白病毒液（对照组），荧光显微镜观察转染24、48、72 h后两组绿色荧光表达情况，72 h时Western blot检测HCN4蛋白表达；电生理检测实验组转染72 h后BMSCs的起搏电流。结果实验组BMSCs 形态正常、生长良好；48 h后荧光显微镜下可见散在的绿色荧光，转染效率约10%；72 h荧光表达稍增多，转染效率为20%～25%。对照组未见绿色荧光表达。Western blot检测示转染72 h后，实验组可见与HCN4蛋白相对分子质量相同的条带表达，而对照组仅有微弱表达；实验组蛋白条带灰度值为33.75 ± 0.41，显著高于对照组的23.39 ± 0.33（t=17.524，P=0.013）。实验组转染后的BMSCs检测到起搏电流，并能被CsCl完全阻断，符合起搏电流特点。结论重组LVs介导HCN4基因成功转染大鼠BMSCs，并检测到HCN4蛋白表达及起搏电流。

引用本文： 于风旭,方易冰,刘炫,赖前成,贾建博,廖斌. 重组慢病毒介导超极化激活的环核苷酸门控阳离子通道4基因转染BMSCs的实验研究. 中国修复重建外科杂志, 2013, 27(12): 1512-1516. doi: 10.7507/1002-1892.20130331 复制

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11.	Später D, Abramczuk MK, Buac K, et al. A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells. Nat Cell Biol, 2013, 15(9): 1098-1106.
12.	Chandler NJ, Greener ID, Tellez JO, et al. Molecular architecture of the human sinus node insights into the function of the cardiac pacemaker. Circulation, 2009, 119(12): 1562-1575.
13.	Choi KH, Park JK, Kim HS, et al. Epigenetic changes of lentiviral transgenes in porcine stem cells derived from embryonic origin. PLoS One, 2013, 8(8): e72184.
14.	Tiscornia G, Singer O, Verma IM, et al. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat Protoc, 2006, 1(1): 234-240.
15.	Lim TT, Geisen C, Hesse M, et al. Lentiviral vector mediated thymidine kinase expression in pluripotent stem cells enables removal of tumorigenic cells. PLoS One, 2013, 8(7): e70543.
16.	贾建博, 方易冰, 卞铁荣, 等. 脂质体介导hHCN2基因转染HEK293细胞的效率研究. 泸州医学院学报, 2012, 35(3): 248-250.
17.	Nong Y, Zhang C, Wei L, et al. In situ investigation of allografted mouse HCN4 gene transfected rat bone marrow mesenchymal stromal cells with the use of patch-clamp recording of ventricular slices. Cytotherapy, 2013, 15(8): 905-919.
18.	El Khoury N, Mathieu S, Marger L, et al. Upregulation of the hyperpolarization activated current increases pacemaker activity of the sinoatrial node and heart rate during pregnancy in mice. Circulation, 2013, 127(20): 2009-2020.
19.	Bosman A, Sartiani L, Spinelli V, et al. Molecular and functional evidence of HCN4 and caveolin-3 interaction during cardiomyocyte differentiation from human embryonic stem cells. Stem Cells Dev, 2013, 22(11): 1717-1727.
20.	Scavone A, Capilupo D, Mazzocchi N, et al. Embryonic stem cell-derived CD166+ precursors develop into fully functional sinoatrial-like cells. Circ Res, 2013, 113(4): 389-398.
21.	Lu W, Yaoming N, Boli R, et al. mHCN4 genetically modified canine mesenchymal stem cells provide biological pacemaking function in complete dogs with atrioventricular block. Pacing Clin Electrophysiol, 2013, 36(9): 1138-1149.
22.	Müller M, Stockmann M, Malan D, et al. Ca2+ activated K channels-new tools to induce cardiac commitment from pluripotent stem cells in mice and men. Stem Cell Rev, 2012, 8(3): 720-740.

1. Senaratne J, Irwin ME, Senaratne MP. Pacemaker longevity: are we getting what we are promised? Pacing Clin Electrophysiol, 2006, 29(10): 1044-1054.
2. Hauser RG, Hayes DL, Kallinen LM, et al. Clinical experience with pacemaker pulse generators and transvenous leads: an 8-year prospective multicenter study. Heart Rhythm, 2007, 4(2): 154-160.
3. Yu FX, Ke JJ, Fu Y, et al. Protective effects of adenosine in rabbit sinoatrial node ischemia-reperfusion model in vivo: control of arrhythmia by hyperpolarization activated cyclic nucleotide-gated (HCN) 4 channels. Mol Biol Rep, 2011, 38(3): 1723-1731.
4. Meng F, Wang K, Aoyama T, et al. Interleukin-17 signaling in inflammatory, Kupffer cells, and hepatic stellate cells exacerbates liver fibrosis in mice. Gastroenterology, 2012, 143(3): 765-776. e1-3.
5. 邓松柏, 佘强. 窦房结自动除极相关离子通道的研究进展. 心血管病学进展, 2008, 29(5): 736-739.
6. Verkerk AO, van Ginneken AC, Wilders R. Pacemaker activity of the human sinoatrial node: role of the hyperpolarization-activated current, I(f). Int J Cardiol, 2009, 132(3): 318-336.
7. Chandler NJ, Greener ID, Tellez JO, et al. Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker. Circulation, 2009, 119(12): 1562-1575.
8. Moosmang S, Biel M, Hofmann F, et al. Diferential distribution of four hyperpolarzation activated cation channels in mouse brainl. Biol Chem, 1999, 380(7-8): 975-980.
9. Rosen MR, Robinson RB, Brink PR, et al. The road to biological pacing. Nat Rev Cardiol, 2011, 8(11): 656-666.
10. Roubille F, Tardif JC. New therapeutic targets in cardiology: heart failure and arrhythmia: HCN channels. Circulation, 2013, 127(19): 1986-1996.
11. Später D, Abramczuk MK, Buac K, et al. A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells. Nat Cell Biol, 2013, 15(9): 1098-1106.
12. Chandler NJ, Greener ID, Tellez JO, et al. Molecular architecture of the human sinus node insights into the function of the cardiac pacemaker. Circulation, 2009, 119(12): 1562-1575.
13. Choi KH, Park JK, Kim HS, et al. Epigenetic changes of lentiviral transgenes in porcine stem cells derived from embryonic origin. PLoS One, 2013, 8(8): e72184.
14. Tiscornia G, Singer O, Verma IM, et al. Design and cloning of lentiviral vectors expressing small interfering RNAs. Nat Protoc, 2006, 1(1): 234-240.
15. Lim TT, Geisen C, Hesse M, et al. Lentiviral vector mediated thymidine kinase expression in pluripotent stem cells enables removal of tumorigenic cells. PLoS One, 2013, 8(7): e70543.
16. 贾建博, 方易冰, 卞铁荣, 等. 脂质体介导hHCN2基因转染HEK293细胞的效率研究. 泸州医学院学报, 2012, 35(3): 248-250.
17. Nong Y, Zhang C, Wei L, et al. In situ investigation of allografted mouse HCN4 gene transfected rat bone marrow mesenchymal stromal cells with the use of patch-clamp recording of ventricular slices. Cytotherapy, 2013, 15(8): 905-919.
18. El Khoury N, Mathieu S, Marger L, et al. Upregulation of the hyperpolarization activated current increases pacemaker activity of the sinoatrial node and heart rate during pregnancy in mice. Circulation, 2013, 127(20): 2009-2020.
19. Bosman A, Sartiani L, Spinelli V, et al. Molecular and functional evidence of HCN4 and caveolin-3 interaction during cardiomyocyte differentiation from human embryonic stem cells. Stem Cells Dev, 2013, 22(11): 1717-1727.
20. Scavone A, Capilupo D, Mazzocchi N, et al. Embryonic stem cell-derived CD166+ precursors develop into fully functional sinoatrial-like cells. Circ Res, 2013, 113(4): 389-398.
21. Lu W, Yaoming N, Boli R, et al. mHCN4 genetically modified canine mesenchymal stem cells provide biological pacemaking function in complete dogs with atrioventricular block. Pacing Clin Electrophysiol, 2013, 36(9): 1138-1149.
22. Müller M, Stockmann M, Malan D, et al. Ca2+ activated K channels-new tools to induce cardiac commitment from pluripotent stem cells in mice and men. Stem Cell Rev, 2012, 8(3): 720-740.

《中国修复重建外科杂志》

重组慢病毒介导超极化激活的环核苷酸门控阳离子通道4基因转染BMSCs的实验研究

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《中国修复重建外科杂志》

重组慢病毒介导超极化激活的环核苷酸门控阳离子通道4基因转染BMSCs的实验研究

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