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张明杰, 李宗泽, 徐 杨, 等.小鼠CD45-/CD31+肺侧缘细胞诱导分化为血管平滑肌细胞的体外培养.四川大学学报(医学版),2017,48(3):363-367
小鼠CD45-/CD31+肺侧缘细胞诱导分化为血管平滑肌细胞的体外培养
Differentiation of Cultivated CD45-/CD31+ Mouse Lung Side Population Cells into Vascular Smooth Muscle
  
中文关键词:  侧缘细胞 血管平滑肌细胞 ABCG2
英文关键词:Side population cells Vascular smooth muscle cells ABCG2
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中文摘要:
      目的探讨在体外诱导分化CD45-/CD31+肺侧缘细胞的特性。方法取小鼠肺组织,流式细胞术分选小鼠CD45-/CD31+肺侧缘细胞;免疫荧光检测其侧缘细胞表型标记物ATP结合盒转运蛋白G2(ABCG2)和干细胞表面标记物干细胞抗原1(stem cell antigen 1,Sca1)的表达,逆转录PCR检测其ABCG2、平滑肌细胞标志物平滑肌肌动蛋白(smooth muscle actin, SMA)及血管平滑肌细胞标志物α-血管平滑肌原肌球蛋白(α-SMT)表达。分选的CD45-/CD31+肺侧缘细胞体外培养14 d后,细胞克隆形成实验和流式细胞术对其进行鉴定。分选的CD45-/CD31+肺侧缘细胞体外诱导14 d后,免疫荧光检测细胞α-SMT的表达,逆转录-PCR检测分化诱导前后细胞中的ABCG2、SMA及α-SMT基因表达。结果成功分选出目的细胞群CD45-/CD31+肺侧缘细胞,其表达ABCG2 和Sca1蛋白,ABCG2和SMA基因,不表达α-SMT基因。细胞体外培养14 d后,出现的细胞克隆鉴定为CD45-/CD31+肺侧缘细胞。CD45-/CD31+肺侧缘细胞分化诱导前表达ABCG2,少量表达SMA,不表达α-SMT,分化诱导14 d后表达α-SMT基因和蛋白、SMA基因,不表达ABCG2基因。结论CD45-/CD31+肺侧缘细胞是血管平滑肌细胞的祖细胞,具有干细胞分化特性。在体外培养可分化为血管平滑肌细胞。
英文摘要:
      Objective To investigate the characteristics of differentiation of lung side population cells (LSP cells)in vitro. Methods CD45-/CD31+ LSP cells sorted by flow cytometry were taken from mouse lung tissues and cultured for 14 d. The cultured LSP cells were observed with colony formation assay and flow cytometryin vitro. The mRNA expressions of ATP-binding cassette transporter G2 (ABCG2), smooth muscle actin (SMA) and α-smooth muscle tropomyosin (α-SMT) in both freshly isolated LSP cells and cultured LSP cells were examined. The expressions of ABCG2 and stem cell antigen 1 (Sca1) in LSP cells were detected using immunofluorescence. RT-PCR tests were performed to detect the expressions of ABCG2, SMA and α-SMT in LSP cells. Results The isolated CD45-/CD31+ lung side population cells expressed ABCG2, SMA and Sca1, but not α-SMT. A large number of LSP in aggregated state were observed after 14 d of culture. Before induction of differentiation, the CD45-/CD31+ LSP cells expressed ABCG2 and SMA, but not α-SMT. After induction of differentiation, the CD45-/CD31+ lung side population cells expressed α-SMT and SMA, but not ABCG2. Conclusion CD45-/CD31+ LSP cells might be progenitor cells of vascular smooth muscle cells,possessing the characteristics of stem cell differentiations.
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