Shox2基因转染后犬骨髓间充质干细胞HCN4表达的变化

罗首鸣; 冯媛媛; 杨攀; 宋治远

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Shox2基因转染后犬骨髓间充质干细胞HCN4表达的变化

论著 | 更新时间：2026-01-09

- Shox2基因转染后犬骨髓间充质干细胞HCN4表达的变化
- Expression profiles of HCN4 in canine mesenchymal stem cells after Shox2 transfection
- 陆军军医大学学报 2015年37卷第9期页码：869-875
- 作者机构：
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  中图分类号： R331.22;R394-33;R394.2
- 纸质出版：2015
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罗首鸣,冯媛媛,杨攀,宋治远.Shox2基因转染后犬骨髓间充质干细胞HCN4表达的变化[J].陆军军医大学学报(原第三军医大学学报),2015,37(09):869-875.

　Luo Shouming,Feng Yuanyuan,Yang Pan,Song Zhiyuan.Expression profiles of HCN4 in canine mesenchymal stem cells after Shox2 transfection[J].J Amry Med Univ (J Third Mil Med Univ),2015,37(09):869-875.
罗首鸣,冯媛媛,杨攀,宋治远.Shox2基因转染后犬骨髓间充质干细胞HCN4表达的变化[J].陆军军医大学学报(原第三军医大学学报),2015,37(09):869-875. DOI：

　Luo Shouming,Feng Yuanyuan,Yang Pan,Song Zhiyuan.Expression profiles of HCN4 in canine mesenchymal stem cells after Shox2 transfection[J].J Amry Med Univ (J Third Mil Med Univ),2015,37(09):869-875. DOI：

摘要

目的探讨矮小同源盒基因亚型2（short stature homobox 2，Shox2）外源基因对犬骨髓间充质干细胞(canine mesenchymal stem cells

cMSCs)体外诱导分化的影响。方法分离培养犬骨髓间充质干细胞，用包装好的两种慢病毒载体pLentis-mShox2-RFP及pLentis-RFP分别转染第3代cMSCs，收集转染阳性的cMSCs与乳鼠心肌细胞(rat ventricular myocytes，NRVMs)共培养。实验分4组：A组（RFP-cMSCs组）、B组（RFP-cMSCs+NRVMs共培养组）、C组（Shox2-RFP-cMSCs组）、D组（Shox2-RFP-cMSCs +NRVMs共培养组）。共培养诱导分化5 d后，利用嘌呤霉素对cMSCs进行纯化，运用全细胞膜片钳检测诱导出内向电流的动力学特征，运用实时荧光定量PCR、Western blot检测HCN4、Tbx3、Cx45/43、Nkx2.5等标志性基因mRNA和蛋白的表达情况，并行免疫荧光检测。结果 Western blot及RT-PCR检测结果显示，D组细胞Shox2、HCN4、Tbx3、Cx45窦房结细胞特异性基因的表达较A、B、C组明显增高（P0.05），B组细胞Cx43及Nkx2.5较C、D组细胞明显增高（P0.05）；免疫荧光结果显示，在Shox2基因调控作用下，cMSCs有HCN4通道生成；膜片钳检测结果显示，C、D组细胞产生了具有明显时间-电压依赖性的内向电流，且对细胞外Cs

敏感，符合起搏离子流I

的动力学特征；而A、B组未检测出此电流。结论经mShox2基因修饰的cMSCs通过体外诱导，产生了起搏离子流I

并高表达窦房结标志性基因，成功向具有一定自主搏动能力的窦房结样细胞分化。

Abstract

Objective To determine the effect of exogenous gene short stature homobox 2 (Shox2) on the differentiation of canine bone marrow mesenchymal (cMSCs) in vitro. Methods cMSCs were isolated by gradient centrifugation

and the cells at passage 3 were

transfected with the lentiviral vector pLentis-mShox2-RFP and pLentis-RFP respectively. Then the cells were divided into 4 groups

that is

the cMSCs transfected with RFP (group A as control)

the cMSCs transfected with RFP and co-cultured with rat ventricular myocytes (NRVMs

group B)

the cMSCs transfected with mShox2-RFP (group C)

and the cMSCs transfected with mShox2-RFP and co-cultured with NRVMs (group D). Patch-clamp technique were introduced to detect the I

current characteristics

while qPCR

Western blotting and immunofluorescence assay were used respectively to test the mRNA and protein expression levels of HCN4

Tbx3

Cx45/43 and Nkx2.5

etc. Results Western blotting and PCR results suggested that the expression of HCN4

Tbx3 and Cx45

mainly expressed in sinus node cells

were significantly higher in the group D than in the other groups (P0.05). Meanwhile

the expression of Cx43 and Nkx2.5 was remarkably higher in the group B than the groups C and D (P0.05). Besides

immunofluorescence assay suggested that Shox2 promoted the generation of HCN4 in the cMSCs. Furthermore

the patch-clamp test indicated that the cMSCs in the groups C and D displayed a hyperpolarization-activated inward current that was highly sensitive to Cs

and showed obviously voltage and time dependences

which met the I

current characteristics. But no such current was found in the cells from groups A and B. Conclusion cMSCs transfected with mShox2 generate I

current and express highly the sinus-node-cell-like genes

which indicating that those cMSCs are successfully differentiated into sinus node-like cells with pacing function.

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