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、100μmol/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(5~50μmol/L)增强EPCs增殖活性(P均<0.01)。50μmol/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。
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 FITCUlex europaeus agglutinin-1(FITC-UEA-1)and the Matrigel tube formation assay were conducted to identify EPCs.The influence of resveratrol at different concentrations(5,10,25,50,100μmol/L)for48 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 48 h,the EPCs’proliferative capacity were improved in concentration(5~50μmol/L)of resveratrol dependence(P<0.01).Compared with the control group,incubated with 50μmol/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.
作者
余惠珍
陈成
陈俊明
朱鹏立
Yu Huizhen;Chen Cheng;Chen Junming;Zhu Pengli(Fujian Provincial Clinical Medical College,Fujian Medical University,Fuzhou 350001,China;The Second Affiliated Hospital of Fujian University of Traditional Chinese Medicine,Fuzhou 350003,China)
出处
《北京中医药大学学报》
CAS
CSCD
北大核心
2020年第1期43-49,共7页
Journal of Beijing University of Traditional Chinese Medicine
基金
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).
关键词
白藜芦醇
内皮祖细胞
增殖
骨髓
大鼠
resveratrol
endothelial progenitor cells
proliferation
bone marrow
rat
