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目的建立人内皮祖细胞(hEPCs)离体培养的方法,探讨培养条件,观察细胞生长状态、形态。xd同时行大鼠EPCs(rEPCs)培养。建立hEPCs和rEPCs培养体系,比较两者不同的生长条件和生长状态。方法:取人脐带血80-120ml/袋,先分离单个核细胞,后使用磁珠细胞分选法(MACS),分选出CD133+/VEGFR2+的细胞,进行流式细胞检测,发现双阳性细胞占48.79%。用差速贴壁法培养5-9天,倒置相差显微镜观察细胞形态并照相、免疫荧光染色后荧光显微镜下观察照相。取大鼠骨髓,冲洗骨髓腔,离心沉淀出细胞,差速贴壁培养法培养、观察。结果:(1)hEPCs在普通光镜下呈索条状、卵圆形。(2)细胞吞噬DiI-acLDL、UEA染料后可在荧光显微镜下特异显色,证明细胞有吞噬功能,推断为hEPCs。(3)CD133+/VEGFR2+的hEPCs细胞,占使用MACS筛选后细胞比例为48.79%。(4)从骨髓中分离出的rEPCs生长活力明显优于hEPCs。结论:(1)该方法培养的hEPCs和rEPCs生长活性好,在普通光镜下呈索条状、卵圆形或铺路石样;(2)hEPCs在细胞数量上可不少于rEPCs,从骨髓中培养出的rEPCs增殖力优于脐血来源的hEPCs。此实验比较并完善了两种不同来源内皮祖细胞培养的方法学及其生长特征,有利于根据不同的细胞培养特性来选择应用于内皮祖细胞的实验研究。
Objective To establish a method for in vitro culture of human endothelial progenitor cells (hEPCs), investigate the culture conditions and observe the cell growth and morphology. xd simultaneous rat EPCs (rEPCs) culture. Establishment of hEPCs and rEPCs culture system, comparing the two different growth conditions and growth status. Methods: Mononuclear cells were isolated from human umbilical cord blood (80-120ml / bag) and then sorted by magnetic bead sorting (MACS). CD133 + / VEGFR2 + cells were sorted out and analyzed by flow cytometry. 48.79%. The cells were cultured for 5-9 days by differential adherence method. The morphology of the cells was observed under inverted phase contrast microscope and photographed under immunofluorescence staining. Bone marrow was taken from rats, the marrow cavity was washed, and the cells were pelleted by centrifugation and cultured by differential adherent culture method. Results: (1) hEPCs showed a strip shape and oval shape under the ordinary light microscope. (2) After phagocytosis of DiI-acLDL and UEA dye, the cells can develop specific color under the fluorescent microscope, which proves that the cells have phagocytic function, which is inferred to be hEPCs. (3) CD133 + / VEGFR2 + hEPCs cells accounted for 48.79% of cells after MACS screening. (4) The rEPCs isolated from bone marrow showed significantly better growth activity than hEPCs. Conclusions: (1) The hEPCs and rEPCs cultured in this method have good growth activity, and are in the shape of strips, oval or paving stones under the ordinary light microscope; (2) hEPCs can be cultured in rEPCs in number of cells, The cultured rEPCs have better proliferative capacity than hEPCs derived from umbilical cord blood. This experiment compares and improves the methodology and growth characteristics of endothelial progenitor cells from two different sources, which is conducive to the choice of experimental studies on endothelial progenitor cells based on different cell culture characteristics.