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目的探讨采用将兔全骨髓直接体外培养诱导分化的方法获取内皮祖细胞(EPCs),同时观察EPCs的扩增能力。方法新西兰兔10只,对每只兔穿刺并抽取骨髓3 ml,用EGM-2培养基对全骨髓进行培养,观察细胞生长及形态变化,绘制细胞生长曲线并评估其扩增能力。对培养12 d后的贴壁细胞行CD133、CD34、VEGFR-2三抗原免疫组化鉴定及吞噬乙酰化低密度脂蛋白(ac-LDL)和结合荆豆凝集素(UEA-1 lectin)的内皮细胞功能鉴定;同时对其行CD133免疫磁珠分选及流式细胞术检测,比较分选前后的CD34+/VEGFR-2+和CD133+/CD34+/VEGFR-2+细胞比例的差异。结果全骨髓直接培养48 h后可见细胞呈丛状或集落样生长,细胞呈梭形、三角形、多边形,细胞生长曲线呈“S”型。经过12 d的培养,每3 ml骨髓可以获得(1.51±0.29)×106个贴壁生长的EPCs,细胞呈铺路石样外观;检测发现CD133、CD34、VEGFR-2三抗原阳性表达,并具有吞噬ac-LDL和结合荆豆凝集素(UEA-1 lectin)的内皮细胞功能。经CD133免疫磁珠分选后CD34+/VEGFR-2+和CD133+/CD34+/VEGFR-2+的细胞比例数分别为分选前的3.38倍和6.14倍。结论通过全骨髓直接培养诱导分化获取兔EPCs的方法简单、可行。
OBJECTIVE: To investigate the feasibility of direct differentiation of EPCs from rabbit bone marrow cells in vitro and to investigate the ability of EPCs to proliferate. Methods Ten New Zealand rabbits were punctured and 3 ml of bone marrow was taken from each rabbit. Whole bone marrow cells were cultured with EGM-2 medium. The growth and morphological changes of cells were observed. The cell growth curve was drawn and its amplification ability was evaluated. Immunofluorescence staining of CD133, CD34 and VEGFR-2 tri-antigens was performed on adherent cells after 12 days of culture. Endothelialization of phagocytosed acetylated low density lipoprotein (ac-LDL) and UEA-1 lectin Meanwhile, CD133 immunomagnetic beads sorting and flow cytometry were performed to detect the difference of the proportion of CD34 + / VEGFR-2 + and CD133 + / CD34 + / VEGFR-2 + cells before and after sorting. Results After cultured for 48 hours, all the cells showed plexiform or colony-like growth. The cells were spindle, triangle and polygon. The cell growth curve showed “S” shape. After 12 days of culture, EPCs (1.51 ± 0.29) × 106 adherent cells were obtained per 3 ml of bone marrow, showing the appearance of paving stones. The positive expression of CD133, CD34 and VEGFR- ac-LDL and endothelial cell binding to UEA-1 lectin. The number of CD34 + / VEGFR-2 + and CD133 + / CD34 + / VEGFR-2 + cells after CD133 immunomagnetic sorting was 3.38 and 6.14 times higher than that before sorting. Conclusion The method of obtaining rabbit EPCs by direct differentiation of whole bone marrow is simple and feasible.