人骨髓内皮祖细胞的分离、诱导培养和扩增  

Isolation,Induction and Culture of Human Endothelial Progenitor Cells in Bone Marrow and Amplification in Vitro

作　　者：柳瑞军[1] 钟竑[1] 单根法[1] 许勤[1] 熊健[1] 

机构地区：[1]上海交通大学医学院附属新华医院胸心外科,上海200092

出　　处：《中国胸心血管外科临床杂志》2008年第2期113-117,共5页Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

基　　金：国家自然科学基金资助项目(30571843)~~

摘　　要：目的探讨并改进从人骨髓中分离、诱导培养和体外扩增内皮祖细胞（EPCs）的方法，为EPCs参与基础研究和临床应用奠定基础。方法采用不同的细胞分离液，用密度梯度离心法从正常人骨髓中分离单个核细胞（hBMMNCs），分别培养在包被人纤维连接蛋白（HFN包被组），包被明胶（明胶包被组）和未包被（未包被组）的培养皿内，利用EBM-2培养4～7d后出现细胞克隆集落（cellcolony—forming units，CFUs），挑选内皮祖细胞样CFUs继续培养（CFUs挑选法），采用流式细胞仪检测CD34＋KDR＋和CD133＋KDR＋双荧光阳性细胞，细胞免疫化学法检测EPCs表面标记CD133、CD34、CD31、vWF和KDR的表达。结果用Optiprep^TM分离液可从人骨髓中更有效地分离出hBMMNCs，进而诱导获取更多EPCs并可进行体外培养扩增；流式细胞仪检测结果显示CD133＋KDR＋细胞高达70．4％±5．4％，CD34＋KDR＋细胞高达69．1％±8．7％；HFN包被组和明胶包被组细胞生长一样旺盛，均可促进EPCs的贴壁生长，促进其生长的作用差异无统计学意义（P〉O．05），而未包被组细胞生长数量最少。培养7d的贴壁细胞表面标记CD133、CD31、vWF和KDR均呈阳性，培养14d的贴壁细胞表面标记CD34呈阳性。结论CFUs挑选法可以从人骨髓中成功地获取更多EPCs并在体外扩增，提出了另一种获取EPCs的高效简单方法，进一步拓宽了获取EPCs的方法和范围。Objective To investigate and improve the method of isolation, induction and culture, amplification in vitro of human endothelial progenitor cells （EPCs） in human bone marrow, thus establish a foundation for EPCs to participate in basic research and clinical application. Methods Human bone marrow mononuclear cells （hBMMNCs） were isolated by density gradient centrifugation and cultured in the 6-plateds coating human fibronections （HFN group）, coating gelatinum （coating gelatinum group） and coating nothing （coating nothing group） respectively. After culturing for 4-7d endothelial cell basal medium-2 （EBM-2） cell colony-forming units （CFUs） appeared, then select EPCs-liked CFUs for cultivation which was named the pick method, CD34＋ KDR＋ and CD133＋ KDR＋ double positive cells were detected in flow cytometry, and CD133, CD34, CD31, vWF and KDR expression were detected with cell immunochemical test. Results hBMMNCs were isolated from human bone marrow more effectively with OptiprepTM cell separating medium, and induced and obtained more EPCs, and cultured and amplificated in vitro. Flow cytometer showed CD133＋ KDR＋ double positive cells reaching up to 70.4% ＋ 5.4%, CD34＋ KDR＋ double positive cells reaching up to 69.1 %± 8.7 %. EPCs grew vigorously in coating HFN group and coating gelatinum group, both HFN and gelatinum promote EPCs adherence and growth, but there were no statistically difference in two groups （P〉0.05）. Surface mark of adherent cells cultured 7d such as CD133, CD31, vWF and KDR showed positive, and cells cultured 14d such as CD34 showed positive. Conclusion The method of picking CFUs can obtain more EPCs from human bone marrow with success and can amplificate EPCs in vitro, thus introducing another simple and effective method to purify EPCs, further widening range and increasing method to purify EPCs.

关 键 词：内皮祖细胞 骨髓 细胞培养 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]