摘要
目的探讨小鼠骨髓内皮祖细胞(EPCs)实用可行的分离、培养及鉴定方法。方法改良密度梯度离心法分离出小鼠骨髓单核细胞,经差速贴壁法筛选后在内皮细胞生长培养基-2MV培养液中培养,倒置显微镜下观察细胞形态,细胞计数试剂盒-8检测细胞增殖力,流式细胞技术鉴定细胞表面标志,管腔形成实验检测其成管腔能力,荧光显微镜检测吞噬Dil标记的乙酰化低密度脂蛋白(Dil—AC—LDL)及异硫氰酸荧光素-荆豆凝集素-1(FITC-UEA-1)及功能。结果初期细胞呈圆形;培养4d细胞后开始转变为短梭形;培养7d呈现集落样生长,数量逐渐增多;培养14d后细胞呈短梭、三角等不同形态;培养21d细胞呈现典型“铺路石”样改变。流式细胞技术检测出:CD34+细胞占84.3%,VEGFR2+细胞占74.1%,而CD45+细胞只占到4.04%,该细胞能在Matrigel基质胶上形成管腔样结构,并具有吞噬DiI—AC—LDL及结合FITC—UEA-1功能。结论通过对前人经验的改良及反复探索,寻找到了一套小鼠骨髓EPCs分离、培养、鉴定的可靠方法,此方法所获得的EPCs增殖能力良好,数量多,生物学特性稳定,可为相关后续研究提供理想的种子细胞。
Objective To explore a practical and feasible method for isolation, culture and identification of mouse bone marrow endothelial progenitor cells(EPCs) . Methods Bone marrow-derived mononuclear cells isolated by density gradient centrifugation were cultured in endothelial cell growth medium-2 MV medium. Growth and morphological changes of the ceils were observed under inverted microscopy. Cell proliferation was observed by cell counting kit-8 assay. Surface markers of the EPCs were detected by flow cytometry. Angiogenic tube formation was determined by Matrigel tube formation assay. Fluores cein isothiocyanat e-ulex europaeus agglutinin-1 (FITC-UEA-1) binding and Dil-Ac-LDL uptake capabilities were observed by fluorescent microscopy. Results In the early stage, the cells were round and spindle-shaped after induced culture for 4 days. After 7 days, the cells grew in colony arrangement and gradually increased in number. After 14 days, the cells were differently shaped, such as short shuttle and triangle. After 21 days, the typical "paving stone" appearance of the cells was observed. The cells were positive for endothelial markers in flow cytometry: CD34 + (84. 3% ), vascular endothelial growth factor receptor 2 + (74. 1% ), but CD45 + (4.04%) . The cells were capable of forming capillary-like tubes, up-taking DiI-Ac-LDL and binding FITC-UEA-1 in Matrigels. Conclusions A reliable method for isolation, cuhure and identifi- cation of mouse bone marrow EPCs may be improved on the basis of previous experiences. Since the EPCs obtained by this method may be capable of good proliferation, large in number, and stable in biological characteristics, they can serve as ideal seed ceils for related subsequent studies.
作者
罗伟
李翔翮
杨先腾
李森磊
王远政
张一
田晓滨
孙立
Luo Wei;Li Xianghe;Yang Xianteng;Li Senlei;Wang Yuanzheng;Zhang Yi;Tian Xiaobin;Sun Li(Graduate School of Guizhou Medical University, Guiyang 550000, Chin;Department of Orthopaedics, People's Hospital of Guizhou Province, Guiyang 550002, China)
出处
《中华创伤骨科杂志》
CAS
CSCD
北大核心
2018年第6期523-528,共6页
Chinese Journal of Orthopaedic Trauma
基金
国家自然科学基金(81560356)
贵州省科学技术基金(黔科合J字[2015]2089号)
贵州省厅社发攻关项目(SY[2015]3044)
关键词
骨髓
细胞培养技术
单核细胞
内皮祖细胞
细胞鉴定
Bone marrow
Cell culture techniques
Monocytes
Endothelial progenitor cells
Cell identification