二甲基亚砜诱导胚胎干细胞分化伴随凋亡发生

Dimethyl sulphoxide induces cell differentiation and apoptosis in mouse embryonic stem cells

目的:标准化二甲基亚砜(DMSO)诱导胚胎干细胞(ESC)向心肌细胞分化的方法,及DMSO是否同时诱导细胞凋亡。方法:MTT法确定DMSO的应用剂量,不同条件培养基对ESC进行诱导分化,并在形态学、蛋白质及基因水平鉴定ESC源心肌细胞。利用形态学、流式细胞仪等对细胞凋亡进行观测。结果:DMSO的最佳应用浓度为1％,拟胚体经诱导后跳动率为96．7％。该心肌细胞表达多种心肌蛋白,且肌小节结构发育成熟。DMSO的促分化效应可能与心肌转录因子GATA-4的表达有关,1％ DMSO可诱导ESC部分产生凋亡,且具有时间和剂量依赖性。结论:1％DMSO不但能够高效诱导ES-D3分化为心肌细胞,且以时间依赖的方式诱导ES-D3细胞部分凋亡。

Objective： To standardize the method by which dimethy sulphoxide （DMSO） induce mouse embryonic stem （ES） cells differentiation into cardiomyocytes, and to investigate whether the apoptosis happens when 1% DMSO was used to induce the differentiation of ES cells. Methods： DMSO was applied to promote the differentiation of ES-D3 cells. The ES-derived cardiomyocytes were identified by the immunocytochemical analysis and the expression of GATA 4 gene was detected by RT-PCR Apoptotic effect was characterized by the morphological change and genome DNA electrophoresis. Apoptotic sub-G1 peak was analyzed by flow cytometry and the expressions of the related apoptotic genes including p53 and bcl-2 were detected by RT-PCR Results： ES-D3 cells successfully differentiated into cardiomyocytes when 1% DMSO was added in the culture medium. The ES-derived cardiomyocytes were characterized by the expressions of cardiac proteins and the ventricular specific gene Myosin Light Chain-2v. 1% DMSO up-regulated GATA-4 expression. DMSO also induced apoptotic change in the ES-D3 cell line at certain concentrations. After 24 h, 1% DMSO induced sub-G1 peak and nucleosomal ladder formation. Up-regulation of p53 rnRNA occurred following 1% DMSO treatment for over 24 h, hut no obvious change was noticed with the expression of bcl-2 mRNA. Conclusion： The administration of 1% DMSO might induce differentiation into cardiomyocytes and apoptosis in the mouse embryonic stem cell line.