解剖学报

解剖学报 ›› 2018, Vol. 49 ›› Issue (4): 443-449.doi: 10.16098/j.issn.0529-1356.2018.04.005

• 细胞和分子生物学 • 上一篇    下一篇

新生大鼠心肌成纤维细胞的内皮细胞分化潜能

张俊悦1 殷国田2 郭志坤1*   

  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.新乡医学院第三附属医院心内科，河南 新乡453003
  • 收稿日期:2017-10-11 修回日期:2018-01-31 出版日期:2018-08-06 发布日期:2018-08-06
  • 通讯作者: 郭志坤 E-mail:1273584012@qq.com
  • 基金资助:
    国家自然科学基金项目

Endothelial differentiation potential of neonatal rat cardiac fibroblasts

ZHANG Jun-yue1 YIN Guo-tian2 GUO Zhi-kun 1*   

  1. 1. He’nan Key Laboratory of Medical Tissue Regeneration; 2.Cardiovascular Department, the Third Affiliated Hospital, Xinxiang Medial University, He’nan Xinxiang453003, China
  • Received:2017-10-11 Revised:2018-01-31 Online:2018-08-06 Published:2018-08-06
  • Contact: GUO Zhi-kun E-mail:1273584012@qq.com

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摘要：

目的  探讨心肌成纤维细胞（CFs）向内皮细胞分化以及成血管潜能。方法  新鲜左心室组织，体外分离、培养、纯化CFs，利用内皮细胞诱导液对第3代 CFs诱导培养，连续诱导培养28 d后，换用内皮细胞培养基（ECM），并消化传代扩增至第3代。观察诱导细胞生长情况和形态变化，利用免疫细胞化学法、流式细胞术和血管形成分析实验对诱导后细胞的内皮细胞标志物的表达和功能特性进行评价。结果 新生大鼠第3代CFs，呈三角形、梭形和多边形，增殖速度迅速；波形蛋白（vimentin）、盘状结构受体2（DDR2）均呈阳性表达。诱导第3天细胞开始汇合，21 d部分细胞形成串珠样连接，28 d出现细胞汇集成环状样形状；免疫组织化学标记vWF和CD31呈阳性表达；细胞免疫荧光标记vWF、CD34、CD105均呈阳性表达；流式细胞术检测诱导后细胞表面标志物CD31表达率为50.5%，对照组CD31表达率为5.82%。结论 血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）体外定向诱导CFs可使其向血管内皮细胞分化，并表现其功能特征。

关键词: 血管内皮生长因子, 碱性成纤维生长因子, 心肌成纤维细胞, 内皮细胞分化, 细胞培养, 大鼠

Abstract:

Objective  To investigate the differentiation of cardiac fibroblasts (CFSs) into endothelial cells and the potential for vascularization, and to provide a cytological and theoretical basis for clinical treatment of myocardial injury. Methods  CFSs were isolated, cultured and purified from the fresh left ventricular tissue. The 3rd generation of CFSs was induced by endothelial cells induced medium for 28 days. After continuous induction culture of 28 days, the endothelial cell medium (ECM) was replaced, digested and passaged down to P3 generation. The growth and morphological changes of the induced cells were detected. The expression of the endothelial cell markers and functional characteristics in the induced cells were evaluated by immunocytochemistry, flow cytometry and angiogenesis analysis. Results  The third generation of CFSs in neonatal rats was triangular, spindle and polygonal, and the rate of proliferation was rapid. Vimentin and discoidin domain receptor 2(DDR2) expressions were positive. The 3rd generation of CFSs was cultured for 3 hours and the induced medium was added, cells began to converge after 3 days, the cells formed string beads were induced at 21 days, and the cells were pooled into the ring shape at 28 days. The immunohistochemistry staining showed that vWF and CD31 were positively expressed in the differentiated cells, but not expressed in the control group. The immunofluorescence staining shows that vWF, CD34 and CD105 were positive in the experimental group, but not in the control group. Flow cytometry analysis showed that the expression rate of CD31 was 50.5% in the induced cells, whereas the expression in the control group was just 5.82%. Conclusion  Cardiac fibroblasts induced with vascular endothelial growth factor(VEGF) and basic fibroblast growth factor(bFGF ) can differentiate into vascular endothelial cells in vitro, and have the characteristics and functions of vascular endothelial cells.

Key words: Vascular endothelial growth factor, Basic fibroblast growth factor(bFGF), Cardiac fibroblast, Endothelial Cell differentiation, Cell culture, Rat