摘要
目的：探索大鼠内皮祖细胞的分离培养，为缺血性疾病细胞移植治疗提供实验方法。方法：采集大鼠骨髓，梯度密度离心法分离单核细胞，内皮细胞培养液M199培养基培养（含20％FCS＋15ng／ml VEGF），种植于提前包埋了纤维连接蛋白的六孔板培养，应用免疫荧光和流式细胞技术鉴定内皮细胞系列标志：CD34、CD31、Flk-1和祖细胞标志CD133。并通过检测其对FITC标记的UED-1的吸附和内吞DiI-acLDL来进行细胞功能学的鉴定。结果：经过梯度密度离心和贴壁法选择的细胞表达内皮细胞特异性抗原CD34、CD31、Flk-1，部分表达CD133。大鼠骨髓单个核细胞经体外诱导分化，培养第5～14天的贴壁细胞不同程度的表达CD34、CD31、Flk-1和CD31。培养第9天流式细胞仪检测其阳性率分别为（40．12±6．28）％、（15.62±4．31）％、（44．05±8．20）％和（76．1±7．20）％，细胞能特异性吸附FITC标记的荆豆凝集素并内吞DiI-acLDL，并且在Matrigel上可以形成毛细血管样。结论：大鼠骨髓可以分离培养内皮祖细胞并能体外扩增，为内皮祖细胞的移植研究奠定了基础。
Objective To examine whether endothelial progenitor cells （EPCs） can be induced and differentiated from rat bone marrow and identified in vitro. Methods The mononuclear cells of rat bone marrow obtained by Ficoll density gradient centrifugation were cultured in medium 199 supplemented with 20% fetal bovine serum, 15ng/ml VEGF in vitro. The expressions of specific antigens （CD133 , CD34, Flk-1 and CDn ） on cell surface were analyzed by immunofluorescence and flow cytometer. The biological functions of endothelial cells were examined by the adsorption of ulex europaeus -agglutinin （UEA） labeled by fluorescein isothiacyanate （FITC） and DiI-Ac-LDL internalization. Results After in vitro reduction and differentiation of mononuclear cells in rat bone marrow, the adhesive cells expressed CD133 , CD34 and Flk-1 at the 5th day during culture. At the 9th day during culture, the expression of CD133 in adhesive cells decreased, and CD31 in- creased. The positive ratio of CD45^＋/CD34^＋ ,CD133^＋ ,Flk-1 ＋ and CD31^＋ were 40. 12 ± 6. 28 % , 15.62± 4. 31% ,44. 05± 8. 20 % and 76. 1 ± 7.20%, respectively. The experiment of FITC labeled UEA adsorption and DiI Ac-LDL internalization were positive at the 9th days during culture. Conclusion The mononuclear cells from rat bone marrow can be differentiated into EPCs under certain condition, which provides the basic for clinical application of EPCs transplantations.
出处
《中国医师杂志》
CAS
2007年第9期1185-1187,共3页
Journal of Chinese Physician
基金
国家“863”计划（2001AA216031）
高等学校博士学科点专项科研基金（20040001133）
关键词
内皮细胞
细胞培养技术
Endothelial cells
Cell culture technique
