人外周血内皮前体细胞体外增殖规律的动态观察及其特性(1)
【摘要】 目的：探讨人外周血内皮前体细胞(endothelial progenitor cells EPCs)体外诱导分化为内皮细胞(ECs)的生长增殖规律及其特性,以期证实人外周血是临床治疗缺血性疾病一个理想的内皮前体细胞来源。方法密度梯度离心提取单个核细胞，接种在人纤连蛋白包被的培养板上，培养于含有促血管生长因子VEGF、b-FGF、IGF、EGF等的内皮生长基质EGM-2 MV中。每天倒置显微镜观察，并记录。4d后去除未附着细胞，继续培养黏附细胞，同时，黏附细胞用Dil-AC-LDL和FITC-UEA-1进行免疫荧光染色，荧光显微镜和共聚焦激光扫描显微镜观察。收集第7d的黏附细胞，流式细胞仪检测细胞表面标志CD34和CD31。结果接种1d后，一些细胞变形；2d后，有细胞团形成；3d后,细胞团周围一些贴壁细胞开始出现；4d后，黏附细胞呈短梭形或多角形贴壁生长，荧光显微镜、共聚焦激光扫描显微镜下可观察到Dil-AC-LDL和FITC-UEA-1双阳性的黏附细胞；第6d、7d，黏附细胞呈长梭形；FACS分析，CD34和CD31阳性率分别为 (14.13±2.79)%、(54.67±3.44)%。结论外周血中确实存在EPCs，并且在促血管生长因子VEGF、b-FGF、IGF、EGF等的刺激下能分化为成熟的内皮细胞。EPCs可望作为临床血管再生的细胞治疗
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关键词：内皮前体细胞；分离；培养；诱导；表面标志; 流式细胞仪
In vitro phenotype maintenance and proliferation of induced endothelial progenitor cells derived from human peripheral blood
【Abstract】Background and Objective：The present aim is to study the isolation，inducement and identification of endothelial progenitor cells(EPCs) from peripheral blood mononuclear cells(PBMCs)，and to probe into its biological activity in vitro for the angiogenesistherapy. Methods：Total mononuclear cells (MNC) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin coated culture dishes，and cultured in EGM-2 MV，Which contains VEGF，bFGF，IGF，EGF. After incubation for 4days, the non-adherent cells were removed, and fresh culture medium was applied. At the same time， adherent cells which were double positive for Dil-AC-LDL-up take and lectin binding by direct fluorescent staining were observed under fluorescent and confocal laser microscope respectively .On the seventh days, Adherent cells were further identified by demonstrating the expression of CD34, and CD31 with flow cytometry.. Results：After 1d in culture , shape changed cells were observed. After 2d in culture, large cell clusters appeared.After 3d in culture, around these clusters some adherent cells were observed. After incubation for 4 days, attached cells grewto short spindle-shaped and double positive staining for Dil-AC-LDL and UEA-1 can be observed under a fluorescent microscope. On the seventh day，FACS analysis ，the adherent cells were positive for CD34 (14.13±2.79)% and CD31(54.67±3.44)%. Conclusion：EPCs exist in the peripheral blood，and can be differentiated into mature endothelial cells( ECs) by the stimulation of VEGF，bFGF， IGF and EGF. EPCs can be an appropriate way of the angiogenesis therapy.
, 百拇医药
[Key word] EPCs；isolation；culture; inducement；surface markers
1997年Asahara 等[1]第一个描述了人外周血存在内皮前体。随后的研究表明，来自骨髓的循环内皮前体细胞(circulating endothelial progenitors CEPs)迁移到生理或病理条件下的新血管形成位点并原位分化成内皮细胞，且能快速和及时地修复损伤的血管[2-4]。血管的损伤可
诱导化学因子，如VEGF、bFGF的释放，促进骨髓来源的EPCs快速募集到损伤位点[5，6]。本文通过人外周血EPCs的分离和培养，探讨了其体外诱导分化的生长增殖规律和特性，以期作为病理条件下提供血管新生潜在源泉的治疗手段。
1材料与方法
1.1材料:
, http://www.100md.com
健康成年人新鲜的外周血20ml(所有健康者都没有CAD倾向)淋巴细胞分离液(Ficoll液，天津市川页生化制品有限公司)；内皮生长介质EGM-2 MV(Clonetics)；人纤连蛋白(HumanFibronectin.Chemicon)；PE标记的CD34单克隆抗体(QIAGEN)；FITC标记的CD31单克隆抗体(DIACLONE)；PE标记的KDR单克隆抗体(Santa Cruz BIOtechnology，InC)；Dil标记的乙酰化的低密度脂蛋白(Dil-AC-LDL Biomedical Technologies Inc)；FITC标记的荆豆凝集素-1(FITC-UEA-1 Vector Laboratories Inc)； 倒置显微镜(OLYMPUS CK2)；荧光显微镜(OLYMPUS BX50))；共聚焦激光扫描显微镜(TCS-SP2)。
[ 下 页 ], 百拇医药(秦宏伟)
, http://www.100md.com
关键词：内皮前体细胞；分离；培养；诱导；表面标志; 流式细胞仪
In vitro phenotype maintenance and proliferation of induced endothelial progenitor cells derived from human peripheral blood
【Abstract】Background and Objective：The present aim is to study the isolation，inducement and identification of endothelial progenitor cells(EPCs) from peripheral blood mononuclear cells(PBMCs)，and to probe into its biological activity in vitro for the angiogenesistherapy. Methods：Total mononuclear cells (MNC) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin coated culture dishes，and cultured in EGM-2 MV，Which contains VEGF，bFGF，IGF，EGF. After incubation for 4days, the non-adherent cells were removed, and fresh culture medium was applied. At the same time， adherent cells which were double positive for Dil-AC-LDL-up take and lectin binding by direct fluorescent staining were observed under fluorescent and confocal laser microscope respectively .On the seventh days, Adherent cells were further identified by demonstrating the expression of CD34, and CD31 with flow cytometry.. Results：After 1d in culture , shape changed cells were observed. After 2d in culture, large cell clusters appeared.After 3d in culture, around these clusters some adherent cells were observed. After incubation for 4 days, attached cells grewto short spindle-shaped and double positive staining for Dil-AC-LDL and UEA-1 can be observed under a fluorescent microscope. On the seventh day，FACS analysis ，the adherent cells were positive for CD34 (14.13±2.79)% and CD31(54.67±3.44)%. Conclusion：EPCs exist in the peripheral blood，and can be differentiated into mature endothelial cells( ECs) by the stimulation of VEGF，bFGF， IGF and EGF. EPCs can be an appropriate way of the angiogenesis therapy.
, 百拇医药
[Key word] EPCs；isolation；culture; inducement；surface markers
1997年Asahara 等[1]第一个描述了人外周血存在内皮前体。随后的研究表明，来自骨髓的循环内皮前体细胞(circulating endothelial progenitors CEPs)迁移到生理或病理条件下的新血管形成位点并原位分化成内皮细胞，且能快速和及时地修复损伤的血管[2-4]。血管的损伤可
诱导化学因子，如VEGF、bFGF的释放，促进骨髓来源的EPCs快速募集到损伤位点[5，6]。本文通过人外周血EPCs的分离和培养，探讨了其体外诱导分化的生长增殖规律和特性，以期作为病理条件下提供血管新生潜在源泉的治疗手段。
1材料与方法
1.1材料:
, http://www.100md.com
健康成年人新鲜的外周血20ml(所有健康者都没有CAD倾向)淋巴细胞分离液(Ficoll液，天津市川页生化制品有限公司)；内皮生长介质EGM-2 MV(Clonetics)；人纤连蛋白(HumanFibronectin.Chemicon)；PE标记的CD34单克隆抗体(QIAGEN)；FITC标记的CD31单克隆抗体(DIACLONE)；PE标记的KDR单克隆抗体(Santa Cruz BIOtechnology，InC)；Dil标记的乙酰化的低密度脂蛋白(Dil-AC-LDL Biomedical Technologies Inc)；FITC标记的荆豆凝集素-1(FITC-UEA-1 Vector Laboratories Inc)； 倒置显微镜(OLYMPUS CK2)；荧光显微镜(OLYMPUS BX50))；共聚焦激光扫描显微镜(TCS-SP2)。
[ 下 页 ], 百拇医药(秦宏伟)