心肌微环境对胎盘间充质干细胞向心肌细胞分化的影响

Myocardial microenvironment on placenta-derived mesenchymal stem cells into myocardial cells differentiation in vitro

目的观察心肌微环境对胎盘间充质干细胞(human placenta-derived mesenchymal stem cells,hPDMSCs)向心肌细胞分化的影响。方法用心肌细胞培养上清液模拟心肌微环境诱导hPDMSCs向心肌细胞分化,同时设置20%DMEM培养的对照组。每天于显微镜下观察细胞形态学变化,每两天台盼蓝拒染法计数细胞,免疫组织化学法检测ɑ-横纹肌肌动蛋白的表达,免疫荧光法检测心肌肌钙蛋白(Icardiac troponin I,cTnI)的表达,RT-PCR法检测心钠素(atrial natriuretic factor,ANF)、β-肌球蛋白重链(beta-myocin heavy chain,β-MHC)mRNA的表达。结果对照组细胞保持较快的增殖速度,不表达ɑ-横纹肌肌动蛋白、cTnⅠ、ANF、β-MHC;心肌细胞培养上清液模拟的心肌微环境组细胞增殖速度较慢,表达ɑ-横纹肌肌动蛋白、cTnⅠ、ANF、β-MHC。结论心肌微环境能够诱导hPDMSCs向心肌细胞分化。

【Objective】 To observe myocardial microenvironment on placenta derived mesenchymal stem cells into myocardial cells differentiation in vitro.【Methods】 The medium which was used to incubate murine fetal cardiomyocytes was collected everyday,we called it conditional medium.We incubated hPDMSCs in conditional medium and 20% DMEM separately.Morphologic characteristics were analyzed by confocal and electron microscopy.Expression of ɑ-sarcomeric actin was analyzed by immunohistochemistry.Expression of cTnI was analyzed by immunohistofluorescence.ANFand β-MHC were detected by reverse transcriptase polymerase chain reaction（RT-PCR）.【Results】 Induced cells significantly slowed down the growth rate.However,uninduced cells still maintained rapidder growth rate.Immunocytochemistry and immunofluorescence revealed that induced cells stained positive for ɑ-sarcomeric actin and cTnI.RT-PCR analysis showed that induced cells expressed ANF and β-MHC.All these cardiomyocyte-specific genes were not expressed in uninduced cells.【Conclusions】 Myocardial microenvironment can induce hPDMSCs differentiated into myocardial cells.