上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

骨形态发生蛋白-2诱导骨髓源性心肌干细胞向心肌细胞分化过程中形态和电生理特性的变化

王新艳1，孙英刚2，杨智昉1   

  1. 1.上海医药高等专科学校解剖生理教研室， 上海 201318； 2.上海交通大学 医学院附属新华医院心血管内科， 上海 200092
  • 出版日期:2013-07-28 发布日期:2013-08-22
  • 通讯作者: 杨智昉, 电子信箱: yangzhifang2011@hotmail.com。
  • 作者简介:王新艳（1978—），女，助教，硕士; 电子信箱: wang_gost@163.com。
  • 基金资助:

    上海高校青年教师资助培养计划

Morphological and electrophysiologic changes during differentiation of marrow-derived cardiac stem cells induced with bone morphogenetic protein-2 towards cardiomyocytes

WANG Xin-yan1, SUN Ying-gang2, YANG Zhi-fang1   

  1. 1.Department of Anatomy and Physiology, Shanghai Institute of Health Sciences, Shanghai 201318, China; 2.Department of Cardiovascular Diseases, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
  • Online:2013-07-28 Published:2013-08-22
  • Supported by:

    Shanghai College Young Teacher Cultivation Foundation

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摘要：

目的 研究骨形态发生蛋白-2 （BMP-2）诱导骨髓源性心肌干细胞（MCSCs）向心肌分化中细胞形态变化、特异性蛋白的表达以及电生理学特征变化。方法 从SD大鼠骨髓中筛选MCSCs，用10 ng/mL BMP-2诱导培养24 h后，换成含10%胎牛血清的DMEM继续培养。相差显微镜下观察细胞的形态变化，免疫细胞化学染色观察诱导后细胞心肌特异性蛋白cTnT的表达。以正常大鼠心室肌细胞为对照，用全细胞膜片钳技术记录诱导后细胞的动作电位（AP）和L型钙电流（ICa-L）。结果 BMP-2诱导后MCSCs的形态和排列方式发生变化。诱导后4周，可见多个细胞相互连接形成肌管样结构，cTnT表达呈现密集的横纹样。诱导后3周，可记录到具有心室肌细胞特点的AP，但平台期不明显;4周细胞的AP出现明显的平台期，类似于成年大鼠心室肌细胞的AP。诱导后3周可记录到ICa-L，但电流值较小;4周细胞ICa-L的电流值增大，激活电位为-40 mV，最大激活电位为0 mV，反转电位为+60 mV，与成年大鼠心室肌细胞的ICa-L相似。结论 BMP-2能够诱导MCSCs向心肌分化，分化后的细胞在形态和电生理学方面具有功能性心肌细胞的特征。

关键词: 骨形态发生蛋白-2, 骨髓源性心肌干细胞, cTnT, 动作电位, L型钙电流, 大鼠

Abstract:

Objective To investigate the morphological changes, expression of cardiac specific protein and electrophysiologic changes of the marrow-derived cardiac stem cells (MCSCs) induced with bone morphogenetic protein-2 (BMP-2) during differentiation towards cardiomyocytes. Methods MCSCs were screened from the bone marrow of SD rats, cultured with 10 ng/mL BMP-2 for 24 h, and treated with DMEM containing 10% fetal bovine serum. The morphological changes of cells were observed with phase contrast microscope, and the expression of myocardium-specific protein cTnT of cells was detected with immunocytochemical staining. The ventricular cardiomyocytes of normal rats were served as controls. The action potential and L-type calcium ion current of cells were recorded with the whole cell patch clamp. Results After induction with BMP-2, the morphology and arrangement of MCSCs changed. Four weeks after induction, a few cells were conjunct to form the myotube-like structures, and cTnT exhibited dense and striation-like expression. Three weeks after induction, the action potential of the cells could be recorded, which was similar to that of the ventricular cardiomyocytes, while the plateau of the action potential of the cells was not obvious. Four weeks after induction, the plateau of the action potential was obvious, which was similar to that of the ventricular cardiomyocyte of adult rats. The L-type calcium ion current of the cells could be recorded three weeks after induction, while it was small. The L-type calcium ion current of the cells was increased four weeks after induction, the depolarizing potential was -40 mV, the peak activated potential was 0 mV, and the reversal potential was +60 mV, the characteristics of which were similar to those of ventricular cardiomyocytes of adult rats. Conclusion BMP-2 may induce MCSCs to differentiate into cardiomyocytes. The differentiated cells have the morphological and electrophysiological characteristics of the mature cardiomyocytes.

Key words: bone morphogenetic protein-2, marrow-derived cardiac stem cells, cTnT, action potential, L-type calcium ion current, rats