大鼠骨髓源性内皮祖细胞的分离培养与鉴定  

Isolation, culture and identification of rat bone marrow-derived endothelial progenitor cells

作　　者：卫肖艳[1] 张莉[1] 林明[2] 陈欢[1] 彭博[1] 彭园园[1] 李富运[1] 洪培馨[1] 范伊凡[1] 

机构地区：[1]北京中医药大学针灸推拿学院,北京市100029 [2]北京大学医学部基础医学院细胞生物学与遗传学系,北京市100083

出　　处：《中国组织工程研究》2013年第14期2570-2577,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金专项基金项目(81141120)~~

摘　　要：背景:内皮祖细胞因其分离与培养的方法各不相同,在实验中难以重复。目的:探讨大量获取骨髓源性内皮祖细胞分离与培养的方法。方法:通过密度梯度离心法从4周龄SD大鼠骨髓中分离单个核细胞,使用EGM-2 MV培养基进行诱导培养,采用形态学特征观察、摄取Dil-Ac-LDL与结合FITC-UEA-1实验、免疫荧光化学鉴定其表面抗原CD133与VEGFR2等方法对其进行鉴定,并通过管腔形成实验观察形成管腔的能力。结果与结论:①形态学观察:分离的骨髓单个核细胞经诱导培养后,在生长的早期(8d左右)、晚期(15d左右)其细胞形态有一定差异,早期以纺锤形、三角形、圆形细胞多见,晚期以圆形、短梭形细胞多见。②摄取Dil-Ac-LDL与结合FITC-UEA-1实验:显示8,21d的细胞均为阳性。③免疫荧光化学染色:8d的细胞表达CD133、VEGFR2。④管腔形成实验:在Matrigel基质上15h左右能够生成血管样结构。结果表明:利用密度梯度离心法分离大鼠骨髓单个核细胞后以EGM-2MV进行诱导培养,经过鉴定证明获得的细胞符合内皮祖细胞的特征。这种方法能够简单、快速、可靠、大量地获取内皮祖细胞。BACKGROUND： Endothelial progenitor cells have a very broad application prospect, but the isolation and culture methods differ greatly and therefore are hard to repeat. OBJECTIVE： To investigate the isolation and culture methods of bone marrow-derived endothelial progenitor ceils. METHODS： Bone marrow mononuclear cells were isolated from 4-week-old Sprague-Dawley rats by density gradient centrifugation methods and cultured by EGM-2 MV medium. Then the cells were identified by morphological observation, FITC-UEA-1 binding and DiI-Ac-LDL uptake assay, and fluorescent immunocytochemistry for detection CD133 and VEGFR2 expression. In addition, angiogenic tube formation was determined by Matrigel tube formation assays. RESULTS AND CONCLUSION： （1） Morphology： After induced culture, the isolated bone marrow mononuclear cells exhibited a spindle-shaped, triangular, and round appearance in the early stage （about the 8th day） and round and short spindle-shaped appearance in the late stage （about the 15th day）. （2） FITC-UEA-1 binding and DU-Ac-LDL uptake assay： Cells were positive on days 8 and 21. （3） Fluorescent immunocytochemistry： on day 8 cells expressed CD133 and VEGFR2. （4） Matdgel tube formation assays： Capillary-like structures formed at 15 hours on Matrigel. These findings suggest that rat bone marrow mononuclear cells isolated by density gradient centrifugation method and cultured by EGM-2MV medium correspond to the characteristics of endothelial progenitor cells. This method is simple, quick, and reliable to harvest enough endothelial progenitor cells.

关 键 词：干细胞 干细胞培养与分化 内皮祖细胞 单个核细胞 密度梯度离心法 细胞培养 细胞鉴定 管腔 形成 国家自然科学基金 干细胞图片文章 

分 类 号：R394.2[医药卫生—医学遗传学]