人脐血CD133^+血管内皮祖细胞的培养、鉴定与分化研究

Study on culture,identification and differentiation of CD133^+ endothelial progenitor cells from human umbilical cord blood

目的探讨从人脐血中分离、体外培养 CD133^+血管内皮祖细胞(EPCs)的方法及其生长特性和鉴定。方法采用密度梯度离心法结合 MACS 分选法提取人脐血中 CD133^+细胞,进行流式细胞仪检测纯度,予 EBM-2培养液接种培养,观察其生长特性;利用 Dil-LDL 及 FITC-Lectin 摄取实验、细胞免疫组织化学等进行鉴定。结果经流式细胞仪检测 CD133^+细胞平均占单核细胞的(1.13±0.10)%,磁珠分选所得 CD133^+细胞平均纯度为(91.45±1.04)%;CD133^+细胞贴壁生长,可分化为梭形血管内皮细胞及形成集落;CD133^+细胞培养过程中 Dil-LDL 及 FITC-Lectin 摄取阳性,双染阳性率为(95.83±1.72)%;CD133^+细胞培养1周后经免疫组织化学检测 CD34、Ⅷ因子阳性率分别为(95.83±2.23)%和(95.92±1.43)%,与人脐静脉内皮细胞比较差异无统计学意义;CD133^+细胞体外培养4d、1周可形成小血管结构。结论免疫磁珠分选法可获取较高纯度 CD133^+血管内皮祖细胞,在生长因子作用下诱导其分化为成熟血管内皮细胞。

Objective To study the isolation, culture and identification of CD133^＋ endothelial progenitor cells （EPCs） from human umbilical cord blood in vitro. Methods EPC separation was performed with density gradient centrifugation and MACS separation. Purity of EPCs was determined by flow cytometry. EPC was cultured with EBM-2 to study the cultivate features of EPC. Uptake test of Dil-LDL and FITC-Lectin and immunohistochemistry were performed. Results According to flow cytometry, （1.13 ± 0. 10）% of mono-nuclear cells were CD133^＋ and the purities of CD133^＋ EPCs were （91.45 ± 1.04） % on average. CD133^＋ EPCs became adherent, spindle-shaped and formed cluster during culture. Uptake test of Dil-LDL and FTTC-Lectin were positive. （95.83 ± 1.72）% of CD133^＋ cells were found positive in both uptake tests. The positive rates of immunostaining of cell markers CD34 and factor ⅤⅢ were （95.83 ±2.23） % and （95.92 ± 1.43） % after cultured for one week, which showed no significant differences between CD133^＋ EPCs and human umbilical vein endothelial cells. Capillary structures were formed by CD133^＋ EPCs after cultured for 4 and 7 d in vitro. Conclusions High purity of CD133^＋ EPCs can be obtained by MACS separation.. CD133^＋ EPCs can differentiate into mature endothelial cells with the effects of stimulating factors.