代谢型谷氨酸受体4在ES细胞体外分化为心肌细胞中的表达研究

Expression of metabotropic glutamate receptor 4 in cardiomyocytes differentiated from mouse embryonic stem cells in vitro

目的:探索代谢型谷氨酸受体4(metabotropic glutamate receptor 4,mGluR4)在小鼠胚胎干细胞(embryonic stem cells,ES细胞)体外分化为心肌细胞中的表达情况。方法:采用悬滴培养法,体外诱导ES细胞分化为心肌细胞,分别以维甲酸(retinoic acid,RA)和溶剂二甲亚砜(dimethyl sulfoxide,DMSO)作为阳性对照和阴性对照;利用免疫荧光法和流式细胞术,鉴定ES细胞衍生心肌细胞团中心肌特异性蛋白α-actinin/Troponin-T和mGluR4共表达情况;用Western Blot法,考察ES细胞定向分化为心肌细胞过程中mGluR4蛋白表达水平;同时观察了小鼠胚胎心脏发育过程中mGluR4基因和蛋白表达变化。结果:ES细胞及其分化来的早期心肌细胞团均表达mGluR4,随着心肌细胞逐渐成熟,mGluR4蛋白表达水平下调。流式细胞术结果显示,贴壁分化11 d的细胞团中mGluR4与心肌特异性蛋白Troponin-T共表达细胞比率仅为(3.00±1.00)%。同时,小鼠胚胎心脏发育过程中mGluR4基因和蛋白表达也呈下降趋势,直至成体小鼠心脏中不表达mGluR4蛋白。结论:ES细胞体外分化为心肌细胞过程中mGluR4有表达并呈下调趋势,与小鼠体内心脏发育过程中mGluR4表达情况基本一致。

Objective： To investigate the expression of metabotropic glutamate receptor 4（mGluR4） in cardiomyocytes differentiated from mouse embryonic stem cells（ES cells）.Methods： ES cells were differentiated into cardiomyocytes with hanging-drop cultures.Retinoic acid（RA） and dimethyl sulfoxide（DMSO） were used as positive and negative controls,respectively.The co-expression of cardiac sarcomeric protein（α-actinin or troponin-T） and mGluR4 were verified by immunocytochemistry and flow cytometry analysis.The mRNA and protein expressions of mGluR4 were verified by RT-PCR and Western blot analysis,respectively.Meanwhile,the expression of mGluR4 in prenatal mouse heart was also examined.Results： mGluR4 was expressed in both mouse ES cells and ES cell-derived cardiomyocytes.The level of mGluR4 protein expression decreased during the maturation of the cardiomyocytes.The co-expression rate of mGluR4 and Troponin T in the beating embryoid bodies（EBs） was only（3.00±1.00）%.On the other hand,mGluR4 gene and protein expressions showed remarkable down-regulation in the development of mouse fetal heart,which was not detected in mouse adult heart.Conclusion： The expression of mGluR4 is down-regulated in the cardiomyocyte differentiation of ES cells.The trend of expression is consistent with that in the prenatal mouse heart development.