微孔法分离大鼠骨髓内皮祖细胞

Isolation of Rat Bone Marrow Derived-Endothelial Progenitor Cells by Micropore-Method

用一种杂交瘤皿,根据内皮祖细胞集落形成单位(endothelial progenitor cells colony-forming units,EPCs-CFUs)的形态特征和EPCs表面特异性标记物分离EPCs.取大鼠股骨、胫骨骨髓,将全骨髓接种在聚苯乙烯制作的杂交瘤皿上,培养4～7天后出现CFUs,将这些集落分别挑选出来后,取单个集落的部分细胞免疫荧光鉴定EPCs表面特异性标记物CD133/VEGFR-2.CD133/VEGFR-2双阳性即为EPCs-CFUs.与此对应的余下一部分继续传代增殖,流式细胞术鉴定CD133/VEGFR-2/CD34,并把此方法命名为微孔法.发现接种后第4天,显微镜下可见明显的CFUs.免疫荧光鉴定大约7%的CFUs为CD133+/VEGFR-2+,进一步传代培养,流式细胞术鉴定CD133+/VEGFR-2+/CD34+细胞纯度达70%以上.传代细胞可在体外形成血管样结构,并表达内皮细胞特异性标记物vWF.结果表明通过微孔法能成功地从大鼠骨髓分离到EPCs.

To explore a new way to purify EPCs, hybridoma dish was used to isolate EPCs from rat bone marrow -derived cells,according to the morphology of endothelial progenitor cells colony-forming units （EPCs-CFUs） and special markers of EPCs. The bone marrow derived cells, from rat femoral bone and shinbone, were plated and cultured on hybridoma dish which was maded of polystyrene. Between 4th and 7th days in culture, stem cells colony-forming units were picked out respectively under microscope. Then one part of this cells was identified by immunofluorescence staining of CD133^＋vEGFR-2^＋, the special markers of EPCs. If the both markers of this part was positive, the rest part of the cells was continuously cultured for passaging. This method was named as ＂Micropore-Method＂ . The conspicuous stem cells colony-forming units were observed under microscope after four days in cluture, about 7% CFUs were CD133^＋/VEGFR-2^＋ positive EPCs-CFUs. After further culturing 7 days, Purity of cells with special markers of CD133^＋/VEGFR-2^＋/CD34^＋ was over 70% identified by flow cytometry. Passaging cells can form capillary tube like formation and differentiate to endothelial like cells expressing EC special marker vWF. It can be concluded that ＂Micropore-Method＂ is a new successful way to isolate EPCs from rat bone marrow.