中国动脉硬化杂志2011，Vol.19Issue(10)：865-869,5.
改良法分离培养兔骨髓源性早晚期内皮祖细胞
Early and Late Endothelial Progenitor Cells Derived from Rabbit Bone Marrow Isolated and Cultured by An Improved Method
摘要
Abstract
Aim To establish an available and convenient method to isolate and culture the rabbit bone marrow-derived endothelial progenitor cells (EPC) and compare the characteristics of the two different EPC. Methods Obtained 2 mL bone marrow from each shinbone of about 4 weeks old New Zealand rabbit, mononuclear cells ( MNC) were I-solated by Ficoll density gradient centrifugation method and planted in the first culture flask, after incubated for 48 h, collecting the suspended cells into the second flask, supplemented with vascular endothelial growth factor ( VEGF) in order to induce cells differentiation into EPC. To compare the growing characteristics of the former and later cells, immunocyto-chemistry was used to examine the expression of the surfaces markers and immunofluorescence for function detection. Results Earlier obtained mononuclear cells adhered after planted 30 minutes, about 3 days late, cells became long spindle-shaped, a little bigger, and formed blood island-like clone and vessels-like formation. To the 10 days, vortex cells can confluence the whole flask, but had less proliferation (early EPC). The second adherent cells, oval shape, and appeared colony-forming by the 5 ~7th day after adhered. At last, cells confluenced like slabstone, which can transfer for 10 generations consecutively (late EPC). The second adherent EPC have a deficiency of CD133 + obviously during differentiation , but a higher expression of CD34 + . Most first adherent EPC can uptake Dil-ac-LDL and Ulex Europaeus agglu-tinin-1 ( UEA-1) , that is similar to the function identification of the second adherent EPC. Conclusion The im-proved Ficoll density gradient centrifugation integrated with differential attachment technique can efficiently isolate and culture rabbit bone marrow-derived EPC, and the late EPC have a stronger growing ability.关键词
内皮祖细胞/单个核细胞/兔骨髓/密度梯度离心法/差速贴壁法Key words
Endothelial Progenitor Cells/Mononuclear Cells/Rabbit Bone Marrow/Ficoll Density Gradient Ultra Centrifugation/Differential Attachment Technique分类
医药卫生引用本文复制引用
王佐,张凯,王仁,苏维,李爽,杨简,姜志胜..改良法分离培养兔骨髓源性早晚期内皮祖细胞[J].中国动脉硬化杂志,2011,19(10):865-869,5.基金项目
国家自然科学基金(81070221)和湖南省教育厅基金(08C747)资助 （81070221）
