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心肌营养素1诱导小鼠诱导性多能干细胞心肌细胞定向分化的实验研究

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2013年第3期
页码:
307-310322
栏目:
基础研究
出版日期:
2013-06-25

文章信息/Info

Title:
Effects of cardiotrophin-1 on cardiac differentiation of mouse induced pluripotent stem cells
作者:
刘 通1; 卜庆婷1; 马 珂1; 金 岩2; 孙冬冬1; 李秀娟1; 曹 丰1
(第四军医大学：1.西京医院心内科，2.组织工程研究中心，陕西 西安 710032)
Author(s):
LIU Tong1;  BU Qingting1;  MA Ke1;  JIN Yan2;  SUN Dongdong1;  LI Xiujuan1;  CAO Feng1
(1.Department of Cardiology, Xijing Hospital, 2.Development Center for Tissue Engineering, Fourth Military Medical University, Xi’an 710032, Shaanxi, China)
关键词:
心肌营养素-1; 小鼠诱导性多能干细胞; 心肌细胞定向分化
Keywords:
cardiotrophin1;  mouse induced pluripotent stem cell;  cardiac differentiation
分类号:
Q254；R542.2
DOI:
-
文献标识码:
A
摘要:
目的:观察心肌营养素-1（CT-1）对小鼠诱导性多能干细胞（miPSCs）心肌细胞定向分化的诱导作用。方法:实验分为对照组与CT1处理组。miPSCs经悬滴法诱导形成拟胚体，第3天培养基中加入1μmol/ml CT-1（CT-1组），对照组采用普通培养基。于第4、7、10和14天，收取细胞样本，采用实时PCR检测心脏特异标志物基因的表达。用免疫荧光染色及流式细胞术检测干细胞标志物Oct4和心肌特异性分化标志物肌钙蛋白I（cTnI）的表达。用透射电镜观察心肌样细胞的超微结构。结果:CT-1组第10天，样本中心肌特异性肌钙蛋白T（cTnT）基因表达的水平为同期对照组的175倍，有显著性差异（P<0.05）。免疫荧光染色法检测显示，两组均有cTnI表达。流式细胞术鉴定的结果显示，对照组与CT1组cTnI的阳性率分别为28.5%和56.4%，有显著性差异（P<0.05）。电镜观察显示，CT-1组肌原纤维及胞内线粒体明显增多，肌纤维排列规则，可见细胞间桥粒连接和缝隙连接。结论:CT-1可显著提高miPSC向心肌细胞定向分化的效率。
Abstract:
AIM:To observe the effect of cardiotrophin1 on cardiac differentiation of mouse induced pluripotent stem cells (miPSCs). METHODS: The differentiation of miPS cells was initiated by embryoid bodies formation with hanging drop method. In experiment group, CT1 application started at day 3 of the differentiation process (1umol/ml, CT1 group) and normal medium was used in control group. Realtime PCR analysis, immunofluorescence staining, flow cytometry analysis and transmission electron microscope were employed to examine the expression of embryonic and cardiac specific cell markers and the cellular ultramicrostructure at day 4, 7, 10 and 14. RESULTS: Compared with that in control group, the expression of cardiac specific marker cTnT in CT1 group at day 10 increased 175 times (P<005). Immunofluorescence staining showed cTnI positive cells in both groups and flow cytometry analysis showed that the rates of cTnI positive cells were 285% and 564%, respectively (P<005). Compared with those in control group, more organized myofibril, mitochondria and cell junction formation were observed in CT1 group. CONCULSION: CT1 significantly promotes the cardiac differentiation of miPS cells.

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备注/Memo

备注/Memo:
收稿日期：2012-12-26.
基金项目：军队十二五重点项目资助（BWS12J037）；国家自然科学基金资助（30970845，81270168）
通讯作者：曹丰，教授，主要从事干细胞分子影像研究Email:wind8828@gmail.com
作者简介：刘通，硕士生Email：liutong525@gmail.com
更新日期/Last Update: 2013-07-16