大鼠骨髓源性内皮祖细胞的分离培养及鉴定

目的: 探讨内皮祖细胞(endothelial progenitor cells，EPCs)分离培养鉴定方法及其生物学特性。方法： 采用梯密度离心法结合差速贴壁法分离获取大鼠骨髓单核细胞，培养于EGM 2 MV SingleQuots内皮细胞培养液中，分别取48 h内贴壁细胞(早期EPCs)和48 h后贴壁细胞(晚期EPCs)，在倒置显微镜下观察细胞形态，应用流式细胞术鉴定EPCs表面抗原标志CD34，CD133和血管内皮生长因子受体 2（VEGFR 2），激光共聚焦检测EPCs吞噬功能，透射电镜观察EPCs内部超微结构。结果： 单核细胞接种后呈小圆形，早期EPCs呈长梭形，晚期以纺锤形为主，培养7 d后有明显干细胞集落；增殖至第6代以后，形态开始逐渐接近于内皮细胞，并出现管腔样结构。内皮祖细胞表面标志CD34，CD133和VEGFR 2均呈阳性表达，能吞噬Dil标记的乙酰化低密度脂蛋白并结合FITC标记的凝集素 1。透射电镜可观察到内皮细胞特征性细胞器Weible Palade小体。结论： 梯密度离心法结合差速贴壁法能够成功地从骨髓中分离、纯化、培养出内皮祖细胞，其增殖能力强，数量充足，生物学特性稳定。

Isolation,culture and identification of rat bone marrow derived endothelial progenitor cells

Objective: To explore the culture, differentiation and identification method and biological characteristics of the rat bone marrow derived endothelial progenitor cells (EPCs)．Methods: We separated the rat bone marrow mononuclear cells using density gradient centrifugation combined with differential sidewall separation, cultured endothelial cell with EGM-2, obtained adherent cells within 48 h (early EPCs) and adherent cells after 48 h(late EPCs), respectively. The cell morphological images were observed under inverted microscope; EPCs surface antigen markers, CD34, CD133 and VEGFR-2 were identified by the flow cytometry, the phagocytosis of EPCs were detected by laser scanning confocal microscope, the internal ultrastructure of EPCs were observed by transmission electron microscope. Results: Mononuclear cells were small round after the first incubation, the late EPCs was given priority to spindle shaped, and there was obvious stem cell colony after culture in 7 days; till the 6th generations of EPCs, the cellular morphology was gradually close to the endothelial cells, appeared the tube cavity structure. CD34, CD133, VEGFR 2 expressed positive, could absorb Dil-acLDL combined with FITC-UEA-1. There was characteristic organelles Weible-Palade (W-P) small body in the EPCs under inverted microscope. Conclusion: The EPC was successfully isolated from bone marrow, and purified, cultured by density gradient centrifugation combined with differential adherent method; it had strong proliferation ability, abundant cell count and stable biological characteristics.  ［Key words］endothelial progenitor cells; bone marrow; stem cells