Gap junction protein-alpha 7 promotes transformation of rat bone marrow mesenchymal stem cells into pacemaker-like cells <italic style="font-style: italic">in vitro</italic>

Yizhang LIN; Chenliang ZHANG; Hua XIAO; Shu LIN; Zhiyuan SONG

doi:10.16016/j.1000-5404.201801028

您当前的位置：

首页 >

文章列表页 >

Gap junction protein-alpha 7 promotes transformation of rat bone marrow mesenchymal stem cells into pacemaker-like cells in vitro

Basic Medicine | 更新时间：2026-01-09

- Gap junction protein-alpha 7 promotes transformation of rat bone marrow mesenchymal stem cells into pacemaker-like cells in vitro
- Journal of Third Military Medical University Vol. 40, Issue 10, Pages: 875-881(2018)
- 作者机构：
  
  400038 重庆，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
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81570300)
- DOI：10.16016/j.1000-5404.201801028
  CLC： R322.11;R329.2;R394.2
- Received：03 January 2018，
  
  Revised：2018-02-03，
  
  Published：30 May 2018
- 稿件说明：
移动端阅览
Yizhang LIN, Chenliang ZHANG, Hua XIAO, et al. Gap junction protein-alpha 7 promotes transformation of rat bone marrow mesenchymal stem cells into pacemaker-like cells in vitro[J]. Journal of Third Military Medical University, 2018, 40(10): 875-881.
DOI：

Yizhang LIN, Chenliang ZHANG, Hua XIAO, et al. Gap junction protein-alpha 7 promotes transformation of rat bone marrow mesenchymal stem cells into pacemaker-like cells in vitro[J]. Journal of Third Military Medical University, 2018, 40(10): 875-881. DOI： 10.16016/j.1000-5404.201801028.

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(I

)。

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(

0.05)；C组Nkx2.5、Cx43则较其他组表达明显升高(

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I

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I

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 I

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 (

0.05)

and the expression levels of Nkx2.5 and Cx43 mRNA were the highest in RFP-transduced rMSCs in the co-culture system (

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 I

current.

Conclusion

GJA7 gene transduction and co-culture with NRVMs can promote the transformation of rMSCs into cardiac pacemaker-like cells capable of generating I

current

which sheds light on a new strategy for pacing therapy with MSCs.

关键词

Keywords

references

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.

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.

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.

GROS D B, JONGSMA H J. Connexins in mammalian heart function[J]. Bioessays, 1996, 18(9): 719-730. DOI: 10.1002/bies.950180907.

WEN L, ZHANG C, NONG Y, et al . Mild electrical pulse current stimulation upregulates S100A4 and promotes cardiog enesis in MSC and cardiac myocytes coculture monolayer[J ] . Cell Biochem Biophys, 2013, 65(1): 43-55. DOI: 10.1007/s12013-012-9402-x.

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.

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.

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.

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.

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.

FENG Y, LUO S, TONG S, et al . Electric-pulse current stimulation increases I f current in mShox2 genetically modified canine mesenchymal stem cells[J ] . Cardiology, 2015, 132(1): 49-57. DOI: 10.1159/000398784..

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.

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.

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.

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..

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.

DESPLANTEZ T, HALLIDAY D, DUPONT E, et al . Cardiac connexinsCx43 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.

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.

KAPOOR N, LIAN G W, MARBÁN 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.

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.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Effects of hyperglycemia and macrophage co-culture on oxidative stress and autophagy in osteoblasts

Role of microfilament depolymerization in osteogenic differentiation of bone marrow mesenchymal stem cells promoted by low intensity pulsed ultrasound

Bone marrow mesenchymal stem cell-derived exosomes promote proliferation, invasion and migration of renal cancer cells by regulating cholestanol metabolism

Effect of BMSCs-derived exosomes on expression of lncRNAs in rat carotid artery after balloon injury

Mechanism of exosomes released from bone marrow stem cells regulating proliferation of cardiac microvascular endothelial cells

Related Author

何忻宜

明叶

谭浩

赵振兴

敬劲

孟雪欢

李想

郑雷蕾

Related Institution

Department of Blood Transfusion, Second Affiliated Hospital, Army Medical University (Third Military Medical University)

Chongqing Maternal and Child Healthcare Hospital

Department of Obstetrics and Gynecology, First Affiliated Hospital, Army Medical Universtity (Third Military Medical University)

Department of Hepatobiliary Surgery, the First Affiliated Hospital of Chongqing Medical University

⁰

Gap junction protein-alpha 7 promotes transformation of rat bone marrow mesenchymal stem cells into pacemaker-like cells in vitro

DOI：10.16016/j.1000-5404.201801028

摘要

Abstract

关键词

Keywords

references