大鼠脾脏内皮祖细胞的分离培养及鉴定

Culture of rat endothelial progenitor cells derived from spleen

目的研究大鼠脾脏内皮祖细胞(EPC)的体外分离、定向培养及鉴定方法。方法用密度梯度离心法获取大鼠脾脏中单个核细胞,用含血管内皮生长因子的DMEM培养液培养,每4天更换培养基去除非贴壁细胞,观察经过不同时间培养后的细胞形态、结构和功能变化。结果培养4 d可发现梭形贴壁细胞,7 d后细胞呈集落状,14 d左右可观察到条索状、网状血管样结构,原代细胞培养21 d左右接近融合并呈典型的鹅卵石样排列。细胞免疫组化显示,培养7 d细胞CD31表达阳性,细胞DiI-LDL摄取和FITC-Lectin结合双阳性,流式细胞仪检测细胞CD133及VEGFR2的阳性率分别为(53.2±3.5)%和(64.5±5.1)%。结论密度梯度离心法联合贴壁筛选及血管内皮生长因子诱导分化可以获得大鼠脾脏EPC。

Objective To culture endothelia1 progenitor cells（EPC）from rat spleen in vitro.Methods Rat spleen cell suspension was prepared by gradient centrifugation of Ficoll and mononuclear cells in the middle layer were collected and then put into DMEM culture medium containing VEGF.The non-adhere cells were excluded every four days by changing the medium,and the morphology,structure and function of the cells were studied.Results The spindle cells were found on the fourth day.On the seventh day,the cluster of cells appeared.The network structure,which was similar with blood vessel,was found 7 days later.The cells looked like ＂cobblestone＂on the 21st day.Immunological cells staining with CD31 was positive.EPC were characterized as adherent cells,double positive for DiI-LDL up take and Lectin binding by direct fluorescent staining.Respectively,the cells were both positive for CD133 and VEGFR2 identified by flow cytometry.Conclusion Density gradient centrifugation combined with adherence cells filtration is an effective way to obtain EPC derived from rat spleen.