内皮祖细胞的体外培养

 目的 建立一个体外培养脐血来源内皮祖细胞（EPC）的培养体系。方法 脐带血经密度梯度离心获得单个核细胞，按本室已建立的培养体系细胞培养，免疫细胞化学和流式细胞术对培养7d后的细胞进行CD34、CD133、vWF、CD146及CD144鉴定。结果 接种后前5d为生长的潜伏期，细胞开始贴壁，无明显扩增。第6天平均每个视野下细胞数目为287+45；第9天细胞数为282+46；第12天开始，细胞进入对数生长期，细胞数为805+67（P<0.05）；第19天细胞继续增殖，细胞数为1115+182（P<0.05）；第23天时，细胞进入凋亡期，数量明显减少，为265+61（P<0.05）。vWF，CD146，CD144表达阳性。流式细胞术结果表明，梭形样细胞群体中，CD34阳性率为88.98%+5.15% (P<0.05),CD133阳性率为1.20%+1.44% (P<0.05)。结论 利用本实验室的培养体系成功培养出内皮祖细胞。

Cultivation of endothelial progenitor cells

Objective To establish a culture system of endothelial progenitor cells from umbilical cord blood. Methods Using density gradient centrifugation to obtain Mononuclear cells,and then cultured them with our labs'culture system.In this study, immunocytochemistry and flow cytometric analysis of CD144,CD146,vWF,CD34 and CD133 were performed. Results The first five days were latent period, cells became adherent, but the number of cells did not increase obviously. At the 6th day, the number of cells came up to 287+45(the average number under 10X10 visual field),and the 9th day up to 282+46（P>0.05）.After cultured for 12 days, the cells came into logarithmic phase ,and the number of cells was 805.33+66.61（P<0.05）,and continued to incease at day 19, the number of cells was up to 1115+182（P<0.05）.Apoptosis took place at day 23, the number of cells decreased to 265+615（P<0.05）. Immunocytochemistric analysis indicated that the cells were weakly positive for vWF,CD144 and CD146, and the percentage of CD34 in cobblestone-shaped cells was 88.98%+5.15%(P<0.05), and that of CD133 was 1.20%+1.44%(P<0.05) with flow cytometric analysis. Conclusions The method established in our lab for EPCs culture is effective. The first five days is a latent period, cells become adherent,but do not increase in number; and from day 12 ,the cells come into logarithmic phase. The number of EPCs is increasing at day 19,and then the cells undergo apoptosis at day 23.