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

林毅漳; 张晨亮; 肖华; 林树; 宋治远

doi:10.16016/j.1000-5404.201801028

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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

基础医学 | 更新时间：2026-01-09

- 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
- Gap junction protein-alpha 7 promotes transformation of rat bone marrow mesenchymal stem cells into pacemaker-like cells in vitro
- 第三军医大学学报 2018年40卷第10期页码：875-881
- 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：
  
  [ 宋治远, E-mail: zysong2010@126.com SONG Zhiyuan, E-mail: zysong2010@126.com
- 基金信息：
  
  国家自然科学基金面上项目(81570300)
- DOI：10.16016/j.1000-5404.201801028
  中图分类号： R322.11;R329.2;R394.2
- 收稿：2018-01-03，
  
  修回：2018-02-03，
  
  纸质出版：2018-05-30
- 稿件说明：
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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.

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.
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. DOI： 10.16016/j.1000-5404.201801028.

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

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