天津医药

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拟胚体高密度培养促进小鼠胚胎干细胞向心肌细胞分化

陈明1,毕琳琳2,赵芳1,王智泉1,干学东1,王扬淦3   

  1. 1. 武汉大学中南医院心内科
    2. 南方医科大学神经生物学教研室
    3. 武汉大学中南医院
  • 收稿日期:2013-01-04 修回日期:2013-03-27 出版日期:2013-08-15 发布日期:2013-08-15
  • 通讯作者: 陈明

High density culture of embryoid bodies enhanced cardiac differentiation of murine embryonic stem cells

CHEN Ming 1,BI Linlin 2,ZHAO Fang 1,WANG Zhiquan 1,GAN Xuedong 1,WANG Yanggan 1   

  1. 1. Department of Cardiology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China
    2. Department of Neurobiology, Southern Medical University
  • Received:2013-01-04 Revised:2013-03-27 Published:2013-08-15 Online:2013-08-15
  • Contact: CHEN Ming

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摘要： 【摘要】 目的 探讨拟胚体（EBs）培养密度对小鼠胚胎干细胞（ESCs）心肌细胞分化的影响。 方法 小鼠 ESCs经过悬滴培养后形成EBs 后，以高、低密度（120、60个EBs/60 mm组织培养皿）贴壁培养， 免疫荧光检测心肌特异性蛋白肌钙蛋白 T(TnT)的表达。 在不同时间点计算不同密度组搏动 EBs 百分比；RT-PCR检测 Nkx2.5、GATA4、β-MHC 及ANF 基因表达水平； Western-blot 检测 TnT及 p38 表达水平。 结果 通过悬滴培养法形成的 EBs 能够出现自发性搏动并且有 TnT 表达。 高密度培养组与低密度培养组相比具有更高的搏动 EBs 百分比， Nkx2.5、GATA4、β-MHC 和ANF 基因高表达水平， 以及 TnT蛋白高表达水平（P＜ 0.05）。 高密度培养组的 p38 活性更高， 使用 p38 特异性阻断剂SB203580能够降低高密度组搏动 EBs 百分比和 TnT蛋白表达水平。 结论 EBs 贴壁培养密度是影响 ESCs 向心肌细胞分化的一个重要因素， EBs 高密度培养相比于低密度培养能够获得更大程度的心肌分化， 且该现象可能是由 p38 信号通路所介导。

关键词: 胚胎干细胞, 细胞计数, 肌细胞, 心脏, 细胞分化, 肌钙蛋白 T, p38 丝裂原活化蛋白激酶类, 拟胚体

Abstract: [Abstract] Objective To investigate the role of different culture densities of embryoid bodies (EBs) in cardiac dif?ferentiation of mouse embryonic stem cells (ESCs). Methods  The mouse ESCs were cultured in hanging drops for 3 days,followed by another 2 days for suspension culture to form EBs. Suspended EBs of different densities (60 or 120 EBs/60 mm tissue culture dish) were transferred onto tissue culture plates. The cardiac specific troponin T (TnT) was detected by immunocytochemistry. The percentage of beating EBs was calculated at different time points. The mRNA expression of cardiac specific transcriptional factors Nkx2.5, GATA4 and cardiac specific proteins β-MHC and ANF were detected by RT-PCR. The protein expressions of TnT and p38 were detected by Western-blot assay. Results    Spontaneously beating EBs were positively stained with TnT. There were significantly higher percentage of beating EBs, higher gene expression levels of Nkx2.5,GATA4, β-MHC and ANF and higher protein expression of TnT in high density culture group than those of low density culture group (P＜ 0.05). Furthermore, there was significantly higher activity of p38 pathway in high density culture group than
that of low density culture group. And the percentages of beating EBs and TnT protein expression were decreased by p38 pathway inhibitor SB203580. Conclusion   The culture density of EBs is important in regulating the cardiac differentiation of ESCs. The high cell-density density culture of EBs enhances the cardiac differentiation of ESCs, which might be mediated by p38 signaling pathway.

Key words: embryonic stem cell, Cell count, myocytes, cardiac, cell differentiation, cell differentiation, p38 mitogen-activated protein kinases, 拟胚体