[1]林毅漳,张晨亮,肖华,等.缝隙连接蛋白α7促进骨髓间充质干细胞转化为心脏起搏样细胞的实验研究[J].第三军医大学学报,2018,40(10):875-881.
 LIN Yizhang,ZHANG Chenliang,XIAO Hua,et al.Gap junction protein-alpha 7 promotes transformation of rat bone marrow mesenchymal stem cells into pacemaker-like cells in vitro[J].J Third Mil Med Univ,2018,40(10):875-881.
点击复制

缝隙连接蛋白α7促进骨髓间充质干细胞转化为心脏起搏样细胞的实验研究(/HTML )
分享到:

《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
40卷
期数:
2018年第10期
页码:
875-881
栏目:
基础医学
出版日期:
2018-05-30

文章信息/Info

Title:
Gap junction protein-alpha 7 promotes transformation of rat bone marrow mesenchymal stem cells into pacemaker-like cells in vitro
作者:
林毅漳张晨亮肖华林树宋治远
陆军军医大学(第三军医大学)第一附属医院心血管内科, 重庆市介入心脏病学研究所
Author(s):
LIN Yizhang ZHANG Chenliang XIAO Hua LIN Shu SONG Zhiyuan

Department of Cardiology, Chongqing Institute of Interventional Cardiology, First Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400038, China
 

关键词:
缝隙连接蛋白&alpha7缝隙连接蛋白骨髓间充质干细胞共培养生物起搏
Keywords:
gap junction protein-alpha 7 connexin bone marrow mesenchymal stem cells co-culture biological pacemaker  
分类号:
R322.11;R329.2;R394.2
文献标志码:
A
摘要:

目的     探讨缝隙连接蛋白α7(GJA7)基因转染大鼠骨髓间充质干细胞(rat mesenchymal stem cells,rMSCs),经与乳鼠心肌细胞(neonatal rat ventricular myocytes,NRVMs)共培养,诱导心脏起搏样细胞相关基因表达的变化。方法    构建携带GJA7基因的慢病毒载体,转染rMSCs,经嘌呤霉素加压筛选纯化后,与NRVMs共培养。分为RFP(red fluorescent protein, 红色荧光蛋白)rMSCs组(A组)、GJA7RFPrMSCs组(B组)、RFPrMSCs+NRVMs组(C组)、GJA7RFPrMSCs+NRVMs组(D组)4组。荧光定量PCR法检测心脏起搏细胞相关基因(HCN4、GJA7、Tbx3、Tbx18)及工作心肌细胞相关基因(Cx43、Nkx2.5)的表达。全细胞膜片钳检测各组细胞起搏电流(If)。结果    携带GJA7基因的慢病毒载体成功转染rMSCs,经嘌呤霉素加压筛选,转染效率高达95%以上。RTqPCR结果显示,D组HCN4、GJA7、Tbx3、Tbx18表达较其他各组明显增高(P<0.05);C组Nkx2.5、Cx43则较其他组表达明显升高(P<0.05)。全细胞膜片钳检测结果显示,仅D组记录到超极化激活的内向电流,该电流具有明显时间电压依赖性,且可被Cscl(4 mmol/L)阻断,符合If电流特性。结论    经GJA7基因修饰并通过与心肌细胞共培养,使rMSCs在体外诱导出具有If电流的心脏起搏样细胞。

Abstract:

Objective    To assess the effect of gap junction protein-alpha 7 (GJA7) gene transduction in promoting the transformation of rat mesenchymal stem cells (rMSCs) into pacemaker-like cells in a co-culture model with neonatal rat ventricular myocytes (NRVMs). Methods    The lentiviral vector carrying both GJA7 gene and red fluorescent protein (RFP) gene (pLentis-GJA7-RFP) or the vector pLentis-RFP were transfected into rMSCs. After screening with puromycin, the transfected rMSCs were cultured alone or in a co-culture system with NRVMs. RT-qPCR was used to detect the mRNA expression levels of cardiac pacemaker cell-related genes (HCN4, GJA7, Tbx3, and Tbx18) and working cardiomyocyte-related genes (Cx43 and Nkx2.5). Whole-cell patch-clamp technique was used to record the pacemaker current If in the cells. Results    The lentiviral vector carrying GJA7 gene  was successfully transfected into rMSCs, and after puromycin screening, the cells showed a transfection efficiency up to 95%. RT-qPCR results showed that the expression of HCN4, GJA7, Tbx3, and Tbx18 mRNA increased significantly in GJA7 gene-transduced rMSCs in the co-culture system as compared with the other cells (P<0.05), and the expression levels of Nkx2.5 and Cx43 mRNA were the highest in RFP-transduced rMSCs in the co-culture system (P<0.05). In GJA7 gene-transduced rMSCs in the co-culture system, but not in the other cells, whole-cell patch clamp recorded a hyperpolarization-activated inward current, which showed a time- and voltage-dependence and could be blocked by Cs+ (4 mmol/L), consistent with the characteristics of If current. Conclusion    GJA7 gene transduction and co-culture with NRVMs can promote the transformation of rMSCs into cardiac pacemaker-like cells capable of generating If current, which sheds light on a new strategy for pacing therapy with MSCs.

参考文献/References:

[1]RIPPLINGER C M, BERS D M. Human biological pacemakers: intrinsic variability and stability[J]. Circulation, 2012, 125(7): 856-858. DOI: 10.1161/CIRCULATIONAHA. 111.086827.
[2]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[J]. Pacing Clin Electrophysiol, 2013, 36(9): 1138-1149. DOI: 10.1111/pace.12154.
[3]DAVIS L M, RODEFELD M E, GREEN K, et al. Gap junction protein phenotypes of the human heart and conduction system[J]. J Cardiovasc Electrophysiol, 1995, 6(10 Pt 1): 813-822. DOI: 10.1111/j.15408167.1995.tb00357. x.
[4]GROS D B, JONGSMA H J. Connexins in mammalian heart function[J]. Bioessays, 1996, 18(9): 719-730. DOI: 10.1002/ bies.950180907.
[5]WEN L, ZHANG C, NONG Y, et al. Mild electrical pulse current stimulation upregulates S100A4 and promotes cardiogenesis in MSC and cardiac myocytes coculture monolayer[J]. Cell Biochem Biophys, 2013, 65(1): 43-55. DOI: 10.1007/s12013-012-9402-x.
[6]MAFI P, HINDOCHA S, MAFI R, et al. Adult mesenchymal stem cells and cell surface characterization  a systematic review of the literature[J]. Open Orthop J, 2011, 5(Suppl 2): 253-260. DOI: 10.2174/1874325001105010253.
[7]HULSMANS M, CLAUSS S, XIAO L, et al. Macrophages facilitate electrical conduction in the heart[J]. Cell, 2017, 169(3): 510-522.e20. DOI: 10.1016/j.cell.2017.03.050.
[8]包馨慧, 李晓梅, 杨毅宁, 等. 原代乳鼠心肌细胞分离培养的改进[J]. 新疆医科大学学报, 2015, 38(5): 564-566,570.DOI: 10.3969/j,issn.10095551.2015.05.010.
BAO X H, LI X M, YANG Y N, et al. An improved protocol for primary cultur of myocytes extracted from neonatal rat [J]. J Xinjiang Med Univ, 2015(5): 564-566,570. DOI: 10.3969/j,issn.1009-5551.2015.05.010.
[9]SIMPSON P, SAVION S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Crossstriations, ultrastructure, and chronotropic response to isoproterenol[J]. Circ Res, 1982, 50(1): 101-116. DOI: 10.1161/01.RES.50.1.101.
[10]JUN C, ZHIHUI Z, LU W, et al. Canine bone marrow mesenchymal stromal cells with lentiviral mHCN4 gene transfer create cardiac pacemakers[J]. Cytotherapy, 2012, 14(5): 529-539. DOI: 10.3109/14653249.2012.654490.
[11]FENG Y, LUO S, TONG S, et al. Electricpulse current stimulation increases If current in mShox2 genetically modified canine mesenchymal stem cells[J]. Cardiology, 2015, 132(1): 49-57. DOI: 10.1159/000398784.
[12]杨绳文,刘志敏. 心脏生物起搏基因治疗的研究进展[J]. 中华心律失常学杂志, 2016, 20(3): 274-276. DOI: 10.3760/cma.j.issn.1007-6638.2016.03.023.
YANG S W, LIU Z M, The road to gene therapy for biological pacemaker [J]. Chin J Cardiac Arrhyth, 2016, 20(3): 274-276. DOI: 10.3760/cma.j.issn.10076638.2016. 03.023.
[13]MAUTINO M R. Lentiviral vectors for gene therapy of HIV-1 infection[J]. Curr Gene Ther, 2002, 2(1): 23-43. DOI: 10. 2174/1566523023348165.
[14]王露, 翟玮玮, 杨向荣, 等. 慢病毒介导RNA干扰沉默Fas基因在脐带间充质干细胞中的表达[J]. 南方医科大学学报, 2014(10): 1475-1480.
WANG L, ZHAI W, YANG X, et al. Lentivirus-mediated stable fas gene silencing in human umbilical cordderived mesenchymal stem cells[J]. J Southern Med Univ, 2014, 34(10): 1475-1480. DOI: 10. 3969/j.issn.1673-4254.2014.10.15.
[15]FUKUHARA S, TOMITA S, YAMASHIRO S, et al. Direct cellcell interaction of cardiomyocytes is key for bone marrow stromal cells to go into cardiac lineage in vitro [J]. J Thoracic Cardiovasc Surg, 2003, 125(6): 1470-1479. DOI: 10.1016/s0022-5223(02)73610-6.
[16]BARUSCOTTI M, BIANCO E, BUCCHI A, et al. Current understanding of the pathophysiological mechanisms responsible for inappropriate sinus tachycardia: role of the if “funny” current[J]. J Interv Card Electrophysiol, 2016, 46(1): 19-28. DOI: 10.1007/s10840-015-0097-y.
[17]DESPLANTEZ T, HALLIDAY D, DUPONT E, et al. Cardiac connexins Cx43 and Cx45: formation of diverse gap junction channels with diverse electrical properties[J]. Pflugers Arch, 2004, 448(4): 363-375. DOI: 10.1007/s00424-004-1250-0.
[18]HOOGAARS W M, ENGEL A, BRONS J F, et al. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria[J]. Genes Dev, 2007, 21(9): 1098-1112. DOI: 10.1101/gad.416007.
[19]KAPOOR N, LIANG W, MARBN E, et al. Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18[J]. Nat Biotechnol, 2013, 31(1): 54-62. DOI: 10.1038/nbt.2465.
[20]ESPINOZA-LEWIS R A, YU L, HE F, et al. Shox2 is essential for the differentiation of cardiac pacemaker cells by repressing Nkx25[J]. Dev Biol, 2009, 327(2): 376-385. DOI: 10.1016/j.ydbio.2008.12.028.

相似文献/References:

[1]曹川,戴霞,李世荣,等.三羟基异黄酮对人瘢痕疙瘩成纤维细胞增殖及侵袭作用的影响[J].第三军医大学学报,2008,30(17):1644.
 CAO Chuan,DAI Xia,LI Shi-rong,et al.Effects of genistein on proliferation and invasion of human keloid fibroblasts[J].J Third Mil Med Univ,2008,30(10):1644.
[2]刘仲荣,张国威,何云志,等.7,12-二甲基苯蒽/巴豆油诱发小鼠皮肤乳头状瘤过程中表皮细胞凋亡的研究[J].第三军医大学学报,2001,23(06):0.[doi:10.16016/j.1000-5404.2001.06.051 ]

更新日期/Last Update: 2018-05-30