摘要
背景:目前组织工程心脏瓣膜再内皮化种子细胞主要来源于成熟内皮细胞,内皮祖细胞作为内皮细胞的前体细胞越来越受到人们的关注。目的:分离和扩增人脐带血内皮祖细胞,观测其体外生物学特性。方法:密度梯度离心法分离新鲜的脐血中单个核细胞,在含血管内皮生长因子和碱性成纤维细胞生长因子的培养液中培养扩增,通过形态学、免疫荧光和流式细胞仪等对贴壁细胞进行鉴定;并与脐静脉内皮细胞进行增殖和迁移能力比较。结果与结论:随着培养和诱导时间延长,贴壁细胞形态发生明显的改变,从小圆形变成梭形,逐渐分化成典型成熟内皮细胞的鹅卵石样形态,并可形成特征性的克隆;体外诱导7d后90%以上贴壁细胞呈Dil-ac-LDL和FITC-UEA-I双阳性;贴壁细胞流式细胞仪分析显示:培养7d的细胞VEGFR-2、CD34和CD133表达分别占(77.4±4.9)%、(52.4±6.6)%和(19.4±2.1)%,培养28d的细胞VEGFR-2和CD34表达分别占(81.1±7.4)%和(7.6±3.1)%,而未检测到CD133表达;人内皮祖细胞增殖和迁移能力明显高于人脐静脉内皮细胞(P<0.05),并且细胞数量可扩增达109L-1。结果显示用密度梯度离心法和贴壁筛选法,可从人脐带血分离、纯化内皮祖细胞;内皮祖细胞可诱导分化为内皮细胞,增殖和迁移能力都很强。
BACKGROUND：At present seeded cells of re-endothelialization for tissue engineered heart valve（TEHV） are mainly from mature endothelial cells;endothelial progenitor cells（EPCs） are precursor cells of endothelial cells,which attracts more and more people＇s attention.OBJECTIVE：To isolate and proliferate human umbilical cord blood-derived（HUCB） endothelial progenitor cells（EPCs）,and to observe their biological characteristics in vitro.METHODS：Mononuclear cells（MNCs） were isolated from fresh umbilical blood by density gradient centrifugation.MNCs were proliferated and cultured in medium supplemented with vascular endothelial grow factor（VEGF） and basic fibroblast growth factor（bFGF）.Attached cells were identified by morphology,immunofluorescence staining and flow cytometry.And the EPCs were compared with human umbilical vein endothelial cells（HUVECs） in the capacity of the proliferation and migration.RESULTS AND CONCLUSION：With the prolongation of culture and induction time,the morphology of attached cells were significantly changed from small round cells to spindle cells,and gradually differentiated into typical mature endothelial cell cobblestone-like morphology,which could form characteristic clone.After 7 days induction in vitro,more than 90% of attached cells presented both Dil-ac-LDL and FITC-UEA-I positive.On day 7,flow cytometric analysis showed that the positive staining rate of attached cells for VEGFR2,CD34 and CD133 were（77.4±4.9）%,（52.4±6.6）%,and（19.4±2.1）%,respectively.On day 28,flow cytometric analysis showed that the positive staining rate of attached cells for VEGFR-2 and CD34 were（81.1±7.4）% and（7.6±3.1）%,but CD133 expression was not detected.The capacity of proliferation and migration of EPCs were significantly higher than that in HUVECs（P 0.05）,and the total number of cells reached 109 /L.The results showed that EPCs can be isolated and purified from HUCB by density gradient centrifugation and adherence screening method.EPCs can be induced and differentiated into endothelial cells,with high capacity of proliferation and migration.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2011年第6期1000-1004,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家"863"高技术研究发展计划(2009AA03Z420)
国家自然科学基金(30872540)
武汉市晨光计划(200950431177)~~
