主 办:北 京 中 医 药 大 学
ISSN 1006-2157 CN 11-3574/R

北京中医药大学学报 ›› 2020, Vol. 43 ›› Issue (1): 43-49.doi: 10.3969/j.issn.1006-2157.2020.01.009

• 中药药理 • 上一篇    下一篇

白藜芦醇对大鼠骨髓内皮祖细胞增殖活性的影响*

余惠珍1, 陈成2, 陈俊明1, 朱鹏立1#   

  1. 1 福建医科大学省立临床医学院 福建 350001;
    2 福建中医药大学附属第二人民医院
  • 收稿日期:2019-08-16 发布日期:2020-02-21
  • 通讯作者: 朱鹏立,男,教授,主任医师,博士生导师,主要研究方向:动脉粥样硬化的基础与临床研究,E-mail:zhupengli7755@163.com
  • 作者简介:余惠珍,女,博士,副教授,主任医师,硕士生导师
  • 基金资助:
    *国家自然科学基金面上资助项目(No.81670258,No.81873515,No.81373785),福建省自然科学基金资助项目(No.2017J01247)

Effects of resveratrol on the proliferation of rat bone marrow-derived endothelial progenitor cells*

Yu Huizhen1, Chen Cheng2, Chen Junming1, Zhu Pengli1#   

  1. 1 Fujian Provincial Clinical Medical College, Fujian Medical University, Fuzhou 350001, China;
    2 The Second Affiliated Hospital of Fujian University of Traditional Chinese Medicine, Fuzhou 350003, China
  • Received:2019-08-16 Published:2020-02-21
  • Contact: Prof. Zhu Pengli, Chief Physician, Doctoral Supervisor. Fujian Provincial Clinical Medical College, Fujian Medical University, Fujian Institute of Clinical Geriatrics, No.134, East Road, Fuzhou 350001, China. E-mail: zhupengli7755@163.com
  • Supported by:
    National Natural Science Foundation of China (No.81670258, No.81873515, No.81373785), Natural Science Foundation of Fujian (No.2017J01247)

摘要: 目的 采用密度梯度离心分离培养大鼠骨髓内皮祖细胞(EPCs),观察白藜芦醇对 EPCs增殖活性的影响。方法 首先采用密度梯度离心法分离大鼠骨髓单个核细胞,去除非贴壁细胞,培养14 d后获得EPCs。随后免疫荧光法测定细胞表面血小板-内皮细胞粘附因子(CD31)、血管性血友病因子(vWF)、血管内皮生长因子受体(FLK-1)、白细胞共同抗原(CD45),行Dil标记的乙酰化低密度脂蛋白(DiI-AC-LDL)和异硫氰酸荧光素荆豆凝集素-1(FITC-UEA-1)双摄取实验,以及基质胶(Matrigel)管状形成实验,鉴定EPCs。运用CCK8法检测细胞增殖活性,采用不同浓度(5、10、25、50、100 μmol/L)白藜芦醇干预EPCs 48 h,以及白藜芦醇在不同时间(24~72 h)干预EPCs,分别检测细胞增殖活性。结果 大鼠骨髓单个核细胞分离后,经诱导培养14 d后,呈现“鹅卵石样”“铺路石状”外观。超过90%细胞表面表达CD31、vWF、FLK-1,能摄取DiI-AC-LDL和结合FITC-UEA-1,可在Matrigel胶中形成管状结构,经上述方法证实,本实验所分离培养的细胞为大鼠EPCs。与对照组比较,干预48 h,白藜芦醇呈浓度依赖性(5~50 μmol/L)增强EPCs增殖活性(P均<0.01)。50 μmol/L白藜芦醇干预EPCs 24 h~72 h后,EPCs的增殖活性呈现时间依赖性增强。结论 采用密度梯度离心法分离培养及免疫荧光法鉴定细胞,证实为大鼠骨髓内皮祖细胞;进一步证实白藜芦醇可增强内皮祖细胞的增殖活性,为后续研究白藜芦醇的心血管保护机制奠定实验基础。

关键词: 白藜芦醇, 内皮祖细胞, 增殖, 骨髓, 大鼠

Abstract: Objective We used isolation of density gradient centrifugation and culture on rat bone marrow-derived endothelial progenitor cells (EPCs) to observe the effect of resveratrol on the proliferative capacity of EPCs. Methods The rat bone marrow-derived mononuclear cells were isolated by using density gradient centrifugation. Nonadherent cells were removed. Adherent cells were cultured for 14 days to obtain EPCs. The cell markers of CD31, vWF, FLK-1, and CD45 were measured by using immunofluorescence. The uptake of Dil-Acetylated Low Density Lipoprotein (DiL-AC-LDL) and FITC-Ulex europaeus agglutinin-1(FITC-UEA-1) and the Matrigel tube formation assay were conducted to identify EPCs. The influence of resveratrol at different concentrations(5, 10, 25, 50, 100 μmol/L)for 48 h and at different time periods (24~72 h) of culture on the proliferative capacity of EPCs were detected by using CCK8 assay. Results Isolated bone marrow derived-mononuclear cells with a cobblestone and pavement stone appearance were cultured after 2 weeks. The immunofluorescence demonstrated that more than 90% cells expressed CD31, vWF and FLK-1. Moreover, the cells could take in DiI-AC-LDL and FITC-UEA-1. In addition, these cells could develop well-formed tube networks when plated in matrigel. According to the morphological, immunophenotypic and functional characteristics, the cells in the study were verified to be rat EPCs. Compared with the control group, after incubated with resveratrol for 48h, the EPCs’ proliferative capacity were improved in concentration (5~50 μmol/L) of resveratrol dependence(P<0.01 ). Compared with the control group, incubated with 50 μmol/L resveratrol for 24, 48 or 72 h, the EPCs’ proliferative capacity were improved in a time (24~72 h)-dependent way(P<0.05,<0.01,<0.01). Conclusion These cells isolated by density gradient centrifugation and detected by immunofluorescence were identified as rat bone marrow-derived endothelial progenitor cells. Furthermore, resveratrol could improve the proliferative capacity of rat bone marrow-derived endothelial progenitor cells. These may establish the foundation for the research in the cardiovascular protection mechanism of resveratrol in the future.

Key words: resveratrol, endothelial progenitor cells, proliferation, bone marrow, rat

中图分类号: 

  • R285.5