骨桥蛋白对骨髓内皮祖细胞体外增殖功能的影响

Effects of osteopontin on proliferation of endothelial progenitor cell in vitro

目的观察骨桥蛋白(OPN)对体外培养的人骨髓源性内皮祖细胞(EPCs)增殖功能的影响及可能的机制。方法以密度梯度离心法获取人骨髓单个核细胞(MNCs),差速贴壁法培养8d,由形态学、流式细胞仪、Dil标记的乙酰化低密度脂蛋白和FITC标记的荆豆凝集素1双染鉴定为EPC后,免疫组化和RT-PCR方法检测OPN在EPC的表达。加入不同浓度人重组OPN和PI3K抑制剂LY294002和Akt抑制剂作用于EPC,采用CCK-8法观测EPC的增殖能力。结果人重组OPN促进EPC的增殖功能,并且呈剂量依赖性。抑制PI3K/Akt信号通路可以阻断OPN对EPC增殖能力的影响。结论人重组OPN在体外可能通过PI3K/Akt信号通路调节人骨髓源性EPC的增殖能力。

Objective To observe the effect of osteopontin（OPN）on the proliferation of endothelial progenitor cells（EPCs）and to explore the underlying mechanisms.Methods The total mononuclear cells（MNCs）were isolated from bone marrow by Ficoll density gradient centrifugation,and EPCs were obtained by differential attachment technique from MNCs after culture for 8 days.The EPCs were identified by morphology observation,flow cytometry and test of the ability to intake the acLDL and bind UEA-1.OPN expression in EPCs was examined by immunohistochemistry and RT-PCR.EPC proliferation was assayed by CCK-8 assay after EPCs were treated with different concentrations of OPN or the inhibitors of phosphoinositide 3-kinase（PI3K）and Akt（LY294002 and Akt inhibitor）.Results The recombinant OPN protein increased EPC proliferation in a dose-dependent manner,and inhibition of the PI3K/Akt pathway blocked the influence of OPN on the proliferation of EPCs.Conclusion Recombinant OPN plays an important role in regulating the in vitro proliferation of bone marrow-derived EPCs via PI3K/Akt pathway.