大鼠骨髓干细胞的分离、培养和诱导生成内皮祖细胞的研究

Isolation,culture and differentiation inducement into endothelia progenitor cell of rat bone marrow stem cell

目的探讨大鼠骨髓干细胞的体外分离、培养和诱导生成内皮祖细胞的的可行性,并检测其表型和功能。方法取大鼠长骨骨髓细胞,第3代细胞传代后加用终质量浓度10μg/L的血管内皮生长因子(VEGF)的细胞因子,培养3 d后行内皮细胞特异性成分鉴定。结果(1)免疫组化染色鉴定:P3代CD133呈中度阳性表现,CD34呈弱阳性表现;经VEGF诱导后表达明显增强。(2)流式细胞仪细胞计数鉴定:P3代CD133、CD34阳性细胞率分别为97.3%、55.5%;经VEGF诱导后CD133、CD34阳性细胞率分别为91.4%、78.6%。(3)P3代VEGF诱导后乙酰化低密度脂蛋白(ac-LDL)、荆豆凝集素(UEA)双染鉴定:经VEGF诱导的P3代细胞ac-LDL和FITC-UEA-1双荧光染色阳性率(70.2±5.1)%,未经VEGF诱导后的P3代细胞ac-LDL和FITC-UEA-1双荧光染色阳性率(20.4±3.8)%。(4)RealTime-PCR检测第3代VEGF cell和三代cell的内皮细胞特异性成分表达:P3 VEGF cell血管内皮生长因子受体2(VEGF-R2)浓度是P3cell的2.22倍。结论用贴壁筛选法和VEGF细胞因子培养大鼠骨髓干细胞可以获得较高纯度的内皮祖细胞(EPCs),该细胞具有内皮祖细胞的特性,可用于进一步向内皮细胞分化的研究与应用。

Objective To investigate the isolation, culture and the differentiation inducement into endothelia progenitor cell of rat bone marrow stem cell. Methods Rat stem cells were obtained from the rat long bone marrow. The stem cells were induced with 10μg/L vascular endothelial growth factor （VEGF） at passage 3 （p3）. Three days after inducement, the endotheliaprogenitor ceils were identified. Results （1）In immunohistochemistry, p3 cells showed moderator CD133 and weak CD34 expression; after the inducement with VEGF, the CD133 and CD34 expression significantly increased. （2）In flow cytometer, the CD133 and CD34 positive cells accounted for 97.3 % and 55.5 % of total Io3 ceils, respectively; after the inducement, the rates of CD133 and CD34 positive cells were 91.4% and 78.6%, respectively. （3） In ac-LDL and FITGUE, A double staining, the positive rate of p3 cells were （20.4 ±3.8）%; after the inducement, the positive rate increased to （70.2 ±5.1）%. （4）In Real-time PCR, the concentration of VEGF-R2 in VEGF induced ceils was 2.2 times as in p3 cells. Conclusion With the adherence screening method and the VEGF inducement, high purified endotheliaprogenitor cells can be obtained.