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目的比较密度梯度离心法及全骨髓培养法分离培养内皮祖细胞的差异。方法取4周雄性近交系C57BL/6J小鼠骨髓,分别使用密度梯度离心法及全骨髓培养法培养,观察细胞贴壁情况和细胞形态,并行DiI-acLDL及FITC-UEA-I双染、vWF、eNOS及细胞表面标志检测。结果密度梯度离心法培养细胞可形成典型铺路石样改变及形成血管样结构;而全骨髓培养法贴壁细胞形态多样,较多呈长梭形铺展生长,部分细胞呈类圆形及纺锤形。比较两种方法培养细胞摄取DiI-acLDL、结合FITC-UEA-I双阳性率以及vWF、eNOS及细胞表面标志表达阳性率,差别均有统计学意义(P<0.05)。应用密度梯度离心法,随着培养时间延长,表达CD34、CD133及FLk-1细胞逐渐增多(P<0.05)。结论密度梯度离心法及全骨髓培养法在EGM-2MV培养体系下均可培养出内皮祖细胞,但密度梯度离心法较全骨髓培养法培养的内皮祖细胞纯度高。
Objective To compare the differences of endothelial progenitor cells isolated by density gradient centrifugation and whole bone marrow culture. Methods The bone marrow of male C57BL / 6J mice was inoculated 4 weeks and cultured with density gradient centrifugation and whole bone marrow culture respectively. The adherent cells and cell morphology were observed. DiI-acLDL and FITC-UEA-I double staining were performed. vWF, eNOS and cell surface markers. Results The cells cultured in density gradient centrifugation could form typical paving stone-like changes and form vascular-like structures. However, the morphology of adherent cells in the whole bone marrow culture varied with long spindle spreading and some cells were round and spindle-shaped. Diagethral acupuncture combined with DiI-acLDL, FITC-UEA-I double positive rate, vWF, eNOS and cell surface marker expression were compared between the two methods (P <0.05). Using density gradient centrifugation, the expression of CD34, CD133 and FLk-1 cells gradually increased with the prolongation of culture time (P <0.05). Conclusions Density gradient centrifugation and whole bone marrow culture can produce endothelial progenitor cells under the culture system of EGM-2MV. However, the density gradient centrifugation method is more efficient than the whole bone marrow culture method.