中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (1): 92-95.doi: 10.3969/j.issn.1673-8225.2011. 01.020

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

悬滴三步法体外诱导小鼠胚胎干细胞向心肌样细胞的分化

刘官信，王芳洁，赵  丹，芦  起   

  1. 重庆医科大学儿童医院儿科研究所，儿童发育疾病研究省部共建教育部重点实验室，  重庆市  400014
  • 收稿日期:2010-08-05 修回日期:2010-11-17 出版日期:2011-01-01 发布日期:2011-01-01
  • 作者简介:刘官信，男，1950年生，重庆市人，汉族，1989年重庆医科大学毕业，副主任技师，主要从事组织细胞培养。 liuguanxin1950@163.com 刘官信和王芳洁为并列第一作者。

Differentiation of mouse embryonic stem cells into cardiomyocytes in hanging drop culture system in vitro  

Liu Guan-xin, Wang Fang-jie, Zhao Dan, Lu Qi   

  1. Institute of Pediatrics, Children’s Hospital, Chongqing Medical University, Key Laboratory of Province and Ministry Coconstruction of Childhood Development Disease Research (Ministry of Education), Chongqing  400014, China
  • Received:2010-08-05 Revised:2010-11-17 Online:2011-01-01 Published:2011-01-01
  • About author:Liu Guan-xin, Associate chief technician, Institute of Pediatrics, Children’s Hospital, Chongqing Medical University, Key Laboratory of Province and Ministry Coconstruction of Childhood Development Disease Research (Ministry of Education), Chongqing 400014, China liuguanxin1950@163.com Liu Guan-xin and Wang Fang-jie contributed equally to the paper.

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542

被引次数

3

摘要：

背景：研究胚胎干细胞分化为心肌细胞的条件和技术，进一步提高诱导分化率、细胞纯度，具有重要的理论和临床应用意义。
目的：建立体外诱导胚胎干细胞分化为心肌细胞模型，为体外研究心肌细胞发育提供合适的实验方法。
方法：采用悬滴-悬浮-贴壁三步法(悬滴三步法)对未分化的胚胎干细胞进行体外诱导分化培养，并按诱导剂的不同分为6组，Ⅰ组：丹参+5-氮杂胞苷；Ⅱ组：丹参+维甲酸；Ⅲ组：5-氮杂胞苷；Ⅳ组：丹参；Ⅴ组：维甲酸；Ⅵ组：阴性对照。诱导剂加入后9 d，免疫细胞化学方法检测拟胚体α-actinin、myosin、α-actin的表达。
结果与结论：诱导剂加入后第2天，倒置显微镜下见拟胚体自发性收缩，每个拟胚体可有1个或多个收缩中心。免疫细胞化学示心肌特异性结构蛋白α-actinin、myosin、α-actin均成阳性表达。Ⅰ组心肌细胞的分化率最高，与Ⅱ、Ⅵ组心肌细胞分化率比较差异有非常显著性意义(P < 0.01)，与Ⅲ、Ⅳ、Ⅴ组比较差异有显著性意义(P < 0.05)。结果显示悬滴三步法联合中药丹参与5-氮杂胞苷为诱导剂，可提高体外胚胎干细胞分化为心肌细胞的分化率。

关键词: 胚胎干细胞, 分化, 心肌细胞, 5-氮杂胞苷, 丹参, 小鼠

Abstract:

BACKGROUND: It is theoretical and clinical significance of studying the condition and technique of the differentiation of embryonic stem cells embryonic stem cell (ESC) into cardiomyocytes and to further elevate rate of differentiation and cell purity.
OBJECTIVE: To set up a model of inducing ESC into cardiomyocytes and to provide a suitable method to study development of cadiomyocytes in vitro. 
METHODS: Undifferentiated ESC were incubated in vitro by hanging drop-suspension-adherence method (hanging drop culture), then divided into six groups according to different inducers added in the culture system: group Ⅰ (salvia miltorrhiza linked with 5-azacytidine as inducer), group Ⅱ (salvia miltorrhiza and retinoic acid as inducer), group Ⅲ (5-azacytidine as inducer), group Ⅳ (salvia miltorrhiza as inducer), group Ⅴ (retinoic acid as inducer), and group Ⅵ (no inducer). After inducer addition, Cardiomyocyte marker proteins expression of α-actinin, myosin, and α-actin were detected by immunocytochemistry in the ninth day. RESULTS AND CONCLUSION: Spontaneously contracting cells appeared in thesecond day following inducer addition under inverted microscope and one embryoid body had one or more contraction foci. The immunocytochemistry showed the cardiomyocyte marker proteins expression of α-actinin, myosin, α-actin were positive. The rate of differentiation of group Ⅰ was highest, which showed significant difference compared with groups Ⅱ and Ⅵ (P  < 0.01), and exhibited significant difference compared with groups Ⅲ, Ⅳ and Ⅴ (P < 0.05). it suggested that traditional Chinese drug salvia miltorrhiza linked with 5-azacytidine as inducers in culture system of hanging drop cultures can improve rates of ESC into cardiomyocytes differentiation in vitro.

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