Electric-Pulse Current Stimulation Increases If Current in mShox2 Genetically Modified Canine Mesenchymal Stem Cells
Electric-Pulse Current Stimulation Increases If Current in mShox2 Genetically Modified Canine Mesenchymal Stem Cells
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DOI：
10.1159/000398784
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发表时间：
2015-01-01
期刊：
影响因子：
1.9
通讯作者：
Song, Zhiyuan
中科院分区：
文献类型：
--
作者：
Feng, Yuanyuan;Luo, Shouming;Song, Zhiyuan
目的：通过电脉冲电流刺激（EPCS）犬间充质干细胞（cMSCs），研究mShox2在体外产生I-f起搏器电流中的作用。方法：采用pLentis-mShox2红色荧光蛋白制备mShox2基因修饰的cMSCs。在EPCS感应后，我们用膜片钳检测了产生的内向电流的动力学特性。然后，我们通过qRT-PCR和Western blotting检测起搏器相关基因Nkx2.5、Tbx3、HCN4、Cx43和Cx45的表达。同时观察形态学变化和心肌分化标志物cTnT。结果：mShox2感染后记录的向内电流与时间和电压相关，为I-f电流。EPCS诱导后，该I-f电流的检出率提高。电流振幅和密度增大，通道激活曲线右移。mShox2感染后，起搏器标志物Tbx3、HCN4和Cx45显著上调，而工作心肌标志物Nkx2.5和Cx43下调，且在EPCS诱导后更为显著。细胞变大，呈纺锤形和蛛形。实验细胞中也检测到cTnT。结论：我们的研究结果表明EPCS促进mShox2基因修饰的cMSCs向起搏器样细胞分化，产生更多的I-f电流。(C) 2015 S. Karger AG，巴塞尔
Objective: We aimed to investigate the role of mShox2 in generating I-f pacemaker current in vitro by means of electric-pulse current stimulation (EPCS) of canine mesenchymal stem cells (cMSCs). Methods: mShox2 genetically modified cMSCs were prepared with pLentis-mShox2 red fluorescent protein. After EPCS induction, we examined the kinetic characteristics of generated inward current by means of a patch clamp. We then evaluated the expression of pacemaker-related genes, such as Nkx2.5, Tbx3, HCN4, Cx43 and Cx45, by means of qRT-PCR and Western blotting. The morphological changes and the cardiomyogenic differentiation marker cTnT were investigated at the same time. Results: The time-and voltage-dependent inward current recorded after mShox2 infection was confirmed to be I-f current. After EPCS induction, the detection rate of this I-f current was increased. The current amplitude and density were increased, and the channel activation curve shifted to the right. The pacemaker markers Tbx3, HCN4 and Cx45 were significantly upregulated, but the working myocardium markers Nkx2.5 and Cx43 were downregulated after mShox2 infection, and were more remarkable after EPCS induction. The cells became larger and assumed spindle and spider-like morphologies. cTnT was also detected in the experimental cells. Conclusions: Our results suggest that EPCS promotes the differentiation of mShox2 genetically modified cMSCs into pacemaker-like cells, which generates more I-f current. (C) 2015 S. Karger AG, Basel