重組慢病毒介導超極化激活的環核苷酸門控陽離子通道4基因轉染BMSCs的實驗研究_《中國修復重建外科雜志》

作者：

于風旭 , 方易冰 , 劉炫 , 賴前成 , 賈建博 , 廖斌

瀘州醫學院附屬醫院心胸外科（四川瀘州，646000）;

關鍵詞：

超極化激活的環核苷酸門控陽離子通道4 BMSCs 慢病毒轉染起搏電流

DOI：

10.7507/1002-1892.20130331

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目的探討重組慢病毒（lentivirus，LVs）介導超極化激活的環核苷酸門控陽離子通道4（hyperpo-larization-activated cyclic nucleotide-gated cation channel 4，HCN4）基因轉染大鼠BMSCs構建生物起搏細胞的可行性。方法選取3～5周齡SD大鼠，采用改良全骨髓貼壁培養法分離培養BMSCs。以LVs作為轉染載體，增強綠色熒光蛋白（enhanced green fluorescent protein，EGFP）為標記，構建LVs-HCN4-EGFP病毒液。取第3代BMSCs分別轉染LVs-HCN4-EGFP病毒液（實驗組）和LVs-EGFP空白病毒液（對照組），熒光顯微鏡觀察轉染24、48、72 h后兩組綠色熒光表達情況，72 h時Western blot檢測HCN4蛋白表達；電生理檢測實驗組轉染72 h后BMSCs的起搏電流。結果實驗組BMSCs 形態正常、生長良好；48 h后熒光顯微鏡下可見散在的綠色熒光，轉染效率約10%；72 h熒光表達稍增多，轉染效率為20%～25%。對照組未見綠色熒光表達。Western blot檢測示轉染72 h后，實驗組可見與HCN4蛋白相對分子質量相同的條帶表達，而對照組僅有微弱表達；實驗組蛋白條帶灰度值為33.75 ± 0.41，顯著高于對照組的23.39 ± 0.33（t=17.524，P=0.013）。實驗組轉染后的BMSCs檢測到起搏電流，并能被CsCl完全阻斷，符合起搏電流特點。結論重組LVs介導HCN4基因成功轉染大鼠BMSCs，并檢測到HCN4蛋白表達及起搏電流。

引用本文： 于風旭,方易冰,劉炫,賴前成,賈建博,廖斌. 重組慢病毒介導超極化激活的環核苷酸門控陽離子通道4基因轉染BMSCs的實驗研究. 中國修復重建外科雜志, 2013, 27(12): 1512-1516. doi: 10.7507/1002-1892.20130331 復制

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5.	鄧松柏, 佘強. 竇房結自動除極相關離子通道的研究進展. 心血管病學進展, 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.
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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.	賈建博, 方易冰, 卞鐵榮, 等. 脂質體介導hHCN2基因轉染HEK293細胞的效率研究. 瀘州醫學院學報, 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. 鄧松柏, 佘強. 竇房結自動除極相關離子通道的研究進展. 心血管病學進展, 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. 賈建博, 方易冰, 卞鐵榮, 等. 脂質體介導hHCN2基因轉染HEK293細胞的效率研究. 瀘州醫學院學報, 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.

《中國修復重建外科雜志》

重組慢病毒介導超極化激活的環核苷酸門控陽離子通道4基因轉染BMSCs的實驗研究

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《中國修復重建外科雜志》

重組慢病毒介導超極化激活的環核苷酸門控陽離子通道4基因轉染BMSCs的實驗研究

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