解剖学报

解剖学报 ›› 2014, Vol. 45 ›› Issue (5): 663-669.doi: 10.3969/j.issn.0529-1356.2014.05.014

• 细胞和分子生物学 • 上一篇    下一篇

肝细胞生长因子和类胰岛素样生长因子对心肌干细胞的诱导作用

朱蓓蓓1,2蔡新华1 孙银平2*   

  1. 1. 新乡医学院河南省医用组织再生重点实验室，河南 新乡 453003; 2. 新乡医学院病理生理学教研室， 河南 新乡 453003
  • 收稿日期:2014-02-10 修回日期:2014-03-24 出版日期:2014-10-06 发布日期:2013-10-06
  • 通讯作者: 孙银平 E-mail:syp2004@xxmu.edu.cn
  • 基金资助:

    新乡医学院研究生科研创新支持计划

Induction effect of hepatocyte growth factor and insulin-like growth factor on cardiac stem cells

ZHU Bei-bei 1,2 CAI Xin-hua1 SUN Yin-ping 2*   

  1. 1. Key Laboratory for Medical Tissue Regeneration of Henan Province, Xinxiang Medical University, He’nan Xinxiang 453003, China; 2. Department of Pathophysiology, Xinxiang Medical University, He’nan Xinxiang 453003, China
  • Received:2014-02-10 Revised:2014-03-24 Online:2014-10-06 Published:2013-10-06
  • Contact: SUN Yin-ping E-mail:syp2004@xxmu.edu.cn

摘要：

目的 探讨肝细胞生长因子（HGF）和类胰岛素样生长因子（IGF1）在外能否诱导心肌干细胞（CSCs）发生增殖并向心肌细胞定向分化。方法 组织贴块法分离培养心肌干细胞，免疫荧光技术鉴定c-kit和CD34表达，流式细胞仪分选纯化c-kit+细胞，CFDA SE荧光探针示踪培养检测细胞增殖特征。实验分为单纯心肌干细胞组和与心肌共培养组，分别用HGF和IGF1干预，倒置显微镜观察细胞不同时期数量与形态的变化，活细胞工作站观察CFDA-SE示踪剂荧光强弱，采集图像，进行统计学分析。免疫荧光技术检测Nkx2.5、心肌肌钙蛋白T（cTnT）的表达。 结果 单纯心肌干细胞组，生长因子刺激后心肌干细胞数量与形态均无明显变化。与心肌共培养组，细胞均发生增殖与形态变化，Nkx2.5、cTnT阳性表达，有个别心肌干细胞分化为自发搏动的心肌细胞。结论 心肌干细胞与心肌细胞共培养条件下，HGF和IGF1能够促进心肌干细胞增殖，联合作用能够诱导心肌干细胞向心肌细胞分化。

关键词: 心肌干细胞, 增殖, 心肌分化, 肝细胞生长因子, 类胰岛素样生长因子, 免疫荧光, 大鼠

Abstract:

Objective To investigate whether hepatocyte growth factor (HGF) and insulin-like growth factor (IGF1) induce cardiac stem cells (CSCs) to proliferate and directly differentiate into cardiomyocytesin vitro. Methods The myocardial tissues were dissected for primary culture of CSCs with the method of explants. The expressions of c-kit and CD34 were examined with immunofluorescence. Primary cells were purified with c-kit by flow cytometry. CFDA SE fluorescent probe was used to detect the proliferation of c-kit+CSCs. C-kit+CSCs were divided into two groups, and cardiac stem cells group and co-cultured with cardiomyocytes group, both group were cultured with HGF and IGF1. An inverted microscope was used to observe changes in cell number and morphology in different periods. Living cells workstation was used to observe CFDA SE fluorescence intensity, to acquire images and do statistical analysis. Immunofluorescence technique was used to detect the expression of Nkx2.5 and cardiac troponin T. Results In cardiac stem cells group，CSCs had no obvious changes in cell number. In co-cultured with cardiomyocytes group, CSCs proliferated and had changes in morphology. Nkx2.5 and cTnT were positively expressed. Several CSCs differentiated into beating cardiomyocytes. Conclusion In co-cultured with cardiomyocytes condition, HGF and IGF1 may promote CSCs to proliferate and differentiate into beating cardiomyocytes.

Key words: Cardiac stem cell, Proliferation, Cardiac differentiation, Hepatocyte growth factor, Insulin-like growth factor, Immunofluorescence, Rat