人和大鼠内皮祖细胞培养及其生长特性的比较  

Culture and identification of Human endothelial progenitor cells and rat endothelial progenitor cells culture

作　　者：高永兴[1] 沈雳[1] 钱菊英[1] 葛均波[1] 

机构地区：[1]上海复旦大学附属中山医院心内科,200032

出　　处：《中国分子心脏病学杂志》2012年第4期233-237,共5页Molecular Cardiology of China

基　　金：国家自然科学基金项目(No.30700318);国家973项目(编号:2010CB529504和2010CB529505)

摘　　要：目的建立人内皮祖细胞(hEPCs)离体培养的方法,探讨培养条件,观察细胞生长状态、形态。xd同时行大鼠EPCs(rEPCs)培养。建立hEPCs和rEPCs培养体系,比较两者不同的生长条件和生长状态。方法:取人脐带血80-120ml/袋,先分离单个核细胞,后使用磁珠细胞分选法(MACS),分选出CD133+/VEGFR2+的细胞,进行流式细胞检测,发现双阳性细胞占48.79%。用差速贴壁法培养5-9天,倒置相差显微镜观察细胞形态并照相、免疫荧光染色后荧光显微镜下观察照相。取大鼠骨髓,冲洗骨髓腔,离心沉淀出细胞,差速贴壁培养法培养、观察。结果:(1)hEPCs在普通光镜下呈索条状、卵圆形。(2)细胞吞噬DiI-acLDL、UEA染料后可在荧光显微镜下特异显色,证明细胞有吞噬功能,推断为hEPCs。(3)CD133+/VEGFR2+的hEPCs细胞,占使用MACS筛选后细胞比例为48.79%。(4)从骨髓中分离出的rEPCs生长活力明显优于hEPCs。结论:(1)该方法培养的hEPCs和rEPCs生长活性好,在普通光镜下呈索条状、卵圆形或铺路石样;(2)hEPCs在细胞数量上可不少于rEPCs,从骨髓中培养出的rEPCs增殖力优于脐血来源的hEPCs。此实验比较并完善了两种不同来源内皮祖细胞培养的方法学及其生长特征,有利于根据不同的细胞培养特性来选择应用于内皮祖细胞的实验研究。Objective To establish a laboratory method of culturing human endothelial progenitor cells （hEPCs） to explore the culture condi- tions, cell growth state, and cellular form. To culture rat endothelial progenitor cells（rEPCs） at the same time. Cell culture conditions and growth state are compared with that of the rat EPCs. Methods The human umbilical cord blood of 80-120ml / bag, firstly wet assorted the mononuclear cells using magnetic bead assisted cell sorting assay, after sub-elect ofCD133 ＋ / VEGFR2＋ cells for flow cytometry, we found that double positive cells accounted for 48.79%. These cells were differentially adherent cultured for 5-9 day and sent to take conventional photography, immunofluorescence staining photography, rEPCs were taken from rat bone marrow by flushing the marrow cavity, and then centrifuged. We adopted the cell differential attachment culture method to culture and observe the cells. Results （1） hEPCs resemble cable strip or oval shape in the light microscope. （2） The sorted cells have phagoeytosis of the DiI-acLDL, UEA dye under fluorescence microscopy and specific color to prove that the cells have a phagocytic function, inferred to be hEPCs.（3） After the use of MACS screening, CD133＋/VEGFR2＋ hEPCs cells accounted for 48.79% of all cells. （4） The growth activity of rEPCs isolated from the bone marrow was significantly superior to that of hEPCs. Conclusions （1） hEPCs and rEPCs cultured in this research are active in growth ,resemble cable strip or oval shape in the light microscope; （2） Proliferation ability of rEPCs from the bone marrow is greater than that of hEPCs from cord blood.This study compared the culture methods and growth characteristics of the two different sources of en- dothelial progenitor cells. The culture method and cell culture characteristics can be used to the experimental study of endothelial progenitor cells for either cell therapy or cellular proliferation and polarization.

关 键 词：内皮祖细胞 培养 DiI-acLDL UEA 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]