不同来源内皮祖细胞培养及其生物学特性比较

Culture of different sources-derived endothelial progenitor cells and comparison of their biological properties

背景:在缺血性疾病中,机体的代偿性反应包括细小动脉形成和血管新生,而内皮祖细胞在其中发挥着重要的作用,参与了机体多种生理、病理性血管重建过程。目前对内皮祖细胞的来源、生物学特性还存在许多争议。目的:分离培养人外周血、脐带血、骨髓以及脐带来源的内皮祖细胞,并对其生物学特性进行比较。方法:采用密度梯度离心法分离人外周血、脐带血以及骨髓单个核细胞进行贴壁培养,脐带来源的内皮祖细胞通过植块贴壁培养或胶原酶消化脐静脉内膜获得的单个核细胞进行贴壁培养。对获得的贴壁细胞进行细胞形态学、增殖活力、细胞周期、免疫表型的检测。结果与结论:①外周血与脐带血来源的贴壁细胞呈长梭形,集落状生长,其上附有成簇圆形细胞,随着培养时间延长,圆形细胞渐脱落,梭形细胞无明显增殖趋势,消化后细胞不能传代;流式显示细胞高表达CD45,部分表达KDR,但不表达CD31。②骨髓和脐带来源的贴壁细胞呈短梭形、多角形;传代后细胞增殖迅速,且细胞形态与增殖活力没有明显下降;多数细胞处于G0/G1期;流式检测显示细胞均不表达CD14、CD45,也不表达CD106、HLA-DR,但高表达CD44、CD90、CD62E、CD73、CD95、CD105;部分表达内皮系标记KDR、vWF;酶消化法获得的脐带内皮祖细胞还表达CD31;共聚焦显微镜扫描显示细胞具有吸收ac-LDL并结合UEA-1的能力。结果说明外周血、脐带血、骨髓与脐带组织中可分离培养出分化程度、增殖活力不同的内皮祖细胞。

BACKGROUND：Compensatory response in ischemic disease included arteriogenesis and angiogenesis.Endothelial progenitor cells （EPCs） play a key role in many physiological and pathological vascular remodeling.There are still controversies in the resource and biological characteristic of EPCs.OBJECTIVE：To isolate EPCs from peripheral blood,umbilical cord blood,bone marrow and umbilical cord,and to compare biological properties of the EPCs.METHODS：Mononuclear cells （MNCs） of peripheral blood,umbilical cord blood,and bone marrow were isolated by density-gradient centrifugation for adherent culture.Umbilical cord-derived EPCs were made into single cell suspension by adherent culture of explants or collagenase digestion of umbilical vein for adherent culture.The obtained adherent cells were detected for cell morphology,proliferation activity,cell cycle and immunophenotype.RESULTS AND CONCLUSION：① Peripheral blood and umbilical cord blood-derived adherent cells grew as clones with spindle shape,and some round cells attached to them.With prolonged culture time,the round cells ablated gradually,and spindle cells had no appear proliferation.After digestion,these cells could not be subcultured.Flow cytometry showed that these cells highly expressed CD45 and partially expressed KDR.But they did not express CD31.②B one marrow-derived cells and umbilical cord-derived adherent cells were spindle and polygonal.Cells proliferated fast,and there was little change in cell morphology and proliferation activity after passage.Most cells were in G0/G1.Flow cytometry showed that these cells expressed neither CD14,CD45,nor CD106,HLA-DR,but highly expressed CD44,CD90,CD62E,CD73,CD95 and CD105.Some expressed endothelial marker：KDR,vWF.Umbilical cord-derived EPCs harvested by enzyme digestion were positive for CD31.Confocal microscope analysis revealed that all these cells could take up ac-LDL and bind UEA-1.Results have suggested that EPCs with different differentiation degrees and proliferation activities could be isolated from peripheral blood,umbilical cord blood,bone marrow and umbilical cord.