大鼠骨髓源性血管内皮祖细胞的体外培养与鉴定  

In vitro culture and identification of endothelial progenitor cells from rat bone marrow

作　　者：邓志锋[1] 鲁友明[1] 汪泱[2] 娄远蕾[2] 郭菲[2] 谢安[2] 

机构地区：[1]南昌大学第二附属医院神经外科,江西省南昌市330006 [2]南昌大学第一附属医院泌尿外科研究所,江西省南昌市330006

出　　处：《中国组织工程研究与临床康复》2008年第16期3069-3073,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30560156);江西省自然科学基金资助项目(0540087)~~

摘　　要：目的：目前内皮祖细胞主要从骨髓、脐带血和外周血获取,但其分离培养方法尚不成熟,外周血来源因损伤小、易获取而被普遍使用,但得到的内皮祖细胞纯度较低。实验拟探讨大鼠骨髓来源内皮祖细胞体外培养、诱导扩增和生物学特性鉴定方法。方法：实验于2006-08/2007-08在南昌大学第一附属医院泌外研究所完成。①实验材料：2周龄SD大鼠由南昌大学医学院动科部提供,雌雄不拘,清洁级,实验过程中对动物处置符合动物伦理学标准。②实验方法：应用梯度离心法采集大鼠骨髓单个核细胞,贴壁筛选法分离内皮祖细胞,置于添加了血管内皮细胞生长因子、人碱性成纤维细胞生长因子、白血病抑制因子、表皮生长因子的M199培养液中扩增培养,对培养2周的细胞采用免疫荧光染色鉴定细胞标志物血管性血友病因子、血管内皮生长因子受体-2、CD34、CD133,采用RT-PCR法检测内皮细胞特异性分子标志血管性血友病因子、FLK-1、Tie-2、CD34、CD133、eNOS基因表达。结果：①培养4d,光电显微镜下可见细胞集落形成,梭形贴壁细胞从集落中央以放射状向外周生长。培养7-10d,细胞集落相互连接,呈典型的＂鹅卵石＂样外观。2周左右可见细胞排列成条索状结构并可呈“微血管样”排列生长。②贴壁细胞免疫荧光染色鉴定细胞标志物血管性血友病因子、FLK-1、CD34、CD133阳性,RT-PCR法检测内皮细胞特异性分子标志血管性血友病因子、FLK-1、Tie-2、CD34、CD133、eNOS基因阳性表达。结论：采用梯度离心法从骨髓中分离单个核细胞,经诱导培养可实现内皮祖细胞的分离培养和体外扩增。AIM： Current isolation and culture methods of endothelial progenitor cells （EPCs） from bone marrow, cord blood and peripheral blood are not mature. Peripheral blood is a common source for EPCs but the obtained cell purity is very low. This study investigated methods of isolating, culturing, differentiation and identification of EPCs from rat bone marrow. METHODS： The experiment was performed at Research Institute of Urinary Surgery, First Hospital of Nanchang University from August 2006 to August 2007. （1）The mononuclear cells were collected from 2-week-old SD rat bone marrow （Nanchang University Medical College, clean grade） by Ficoll density gradient centrifugation. EPCs were isolated with adherence screening method and cultured in M 199 culture medium with the supplement of human basic fibroblast growth factor, leukaemia inhibitory factor, and epidermal growth factor. After 2 weeks of culture, immunofluorescence was used to identify cell markers such as yon Willebrand disease factor, vascular endothelial growth factor receptor-2, CD34, and CD133; RT-PCR was employed to identify cell specific molecular markers including von Willebrand disease factor, FLK- 1, Tie-2, CD34, CD 133, and eNOS gene expression. RESULTS： （1）After four days of culture, cell colony was observed using light electron microscopy; adherent cells showed fusiform and radiated from the centre to the outside of the colony. While cultured for 7 days to 10 days, cell colonies were interconnected with the shape of cobblestone. After 2 weeks of culture, cells arranged in strands and capillary vessel network. （2）Adherent cells were identified positive for yon Willebrand disease factor, FLK-1, CD34, and CD133 by immunofluorescence staining, and positive for yon Willebrand disease factor, FLK-1, Tie-2, CD34, CD133, and eNOS gene expression by RT-PCR. CONCLUSION： EPCs are isolated from bone marrow-derived mononuclear cells, and amplified in vitro.

关 键 词：内皮祖细胞 骨髓源性 体外培养 

分 类 号：R392.2[医药卫生—免疫学]