两种不同内皮祖细胞的分离培养与鉴定

目的:探讨从小鼠骨髓中分离、培养、诱导分化及鉴定两种内皮祖细胞的方法,为进一步研究和临床应用奠定基础。方法:密度梯度离心法分离小鼠骨髓单个核细胞,接种于内皮祖细胞条件培养基,通过贴壁培养法培养出早期内皮祖细胞和晚期内皮祖细胞,并在0 d、6 d、10 d流式鉴定早期内皮祖细胞,在第8周流式鉴定晚期内皮祖细胞。结果:通过体外贴壁扩增培养,从小鼠骨髓细胞中成功培养出EEPC(早期内皮祖细胞)和EOC(晚期内皮祖细胞),表达CD34+/CD133+/VEGFR2+的EEPC比例从最初的0.08%能够增长至70%;EOC大约出现于3-4周,5-8周时呈现指数增长,具有典型的内皮细胞鹅卵石样形态,表达CD31、VEGFR2等内皮细胞表面标志而不表达CD34、CD133等干细胞表面标志。结论:确立了内皮祖细胞体外分离培养和诱导分化的实验方法,为进一步研究奠定基础。

Isolation and Identification of Two Different Kinds of Endothelial Progenitor Cells

Objective:To investigate an appropriate method for isolating and assessing EPCs from mice bone marrow. This could offer a better support to further research on EPCs.Methods:BMmononuclear cells were isolated frommice bone marrow by density gradient centrifugation and were cultured in conditioned medium. Early endothelial progenitor cells and late endothelial progenitor cells were cultured by adherent culture method. Early EPCs were analyzed by FACS on days 0, 6 and 10. EOCs were analyzed after 8 weeks.Results:EEPCs and EOCs were cultured via the adherent culture of bone marrow, the percentage with EEPC phenotypes (CD34+/CD133+/VEGFR2+), increased from 0.08 %to 70 %when cultured for 7 days in the EPC medium. EOC appeared in about 3 to 4 weeks, in 5 to 8 weeks showed exponential growth, which expressed CD31 and VEGFR2.Conclusion:The primary establishment of isolation, cultivation and identification of EPCs frommice bone marrow, indicates that EPCs can be isolated and identified in vitro. It will be the basis for further research and clinical applications.