人脐血中分离培养扩增内皮集落形成细胞及其生物相容性

Culture,large-scale expansion and biocompatibility of endothelial colony forming cells

背景:内皮集落形成细胞是内皮祖细胞的一种亚型,体外培养扩增及其在组织工程中的应用尚不明确。目的:探索从人脐血中分离培养扩增内皮集落形成细胞并观察其生物相容性。方法:采用密度梯度离心法从人脐血中分离单个核细胞,接种于Ⅰ型鼠尾胶原包被的培养板上,采用内皮祖细胞专用EGM-2培养基诱导培养,早期传代后扩增,免疫荧光鉴定细胞,并检测其体外成血管和增殖能力,与纳米羟基磷灰石/磷酸三钙材料复合培养后的生物相容性。结果与结论:脐血来源内皮集落形成细胞呈铺路石样集落生长,吞噬Dil-Ac-LDL并结合FITC-UEA-1,表达CD31,vWF,KDR和Tie-2,早期传代后拥有较强增殖潜能,可在体外大量扩增,在体外基质胶上可形成闭合管腔样结构,复合纳米羟基磷灰石/磷酸三钙材料后生物学特性不受影响。提示可从脐血中分离并体外大量扩增内皮集落形成细胞。

BACKGROUND： There is still uncertain about in vitro culture and expansion of endothelial colony forming cells （ECFCs） and its application in tissue engineering. OBJECTIVE： To elucidate a method for in vitro culture, identification and large-scale expansion of ECFCs and to investigate its biocompatibility with biological material. METHODS： Monocytes were isolated by Ficoll density gradient centrifugation from human umbilical cord blood, in vitro cultured and expanded in EGM-2 medium on tissue culture plates precoated with type I rat tail collagen. Then cells were characterized by immunofluorescence staining. The growth kinetics, tube formation capacity and biocompatibility with nano-hydroxyapatite/tricalcium phosphate （HA/TCP） were observed. RESULTS AND CONCLUSION： During culture, the cells displayed cobble-stone morphology with outgrowth, took up DiI-Ac-LDL, bound FITC-UEA-1, and stained positively for cell markers CD31, vWF, KDR and Tie-2. These cells demonstrated high proliferation rate after passage, formed closed network structures on Matrigel and the bioactivity remained stable when co-cultured with HA/TCP. Results indicate it is feasible to isolate and large-scale expand ECFCs from human umbilical cord blood in vitro and ECFCs may serve as seed cells in tissue engineering.