摘要
目的建立一种有效的标记兔骨髓间充质干细胞（BMSCs）的方法。方法2周龄新西兰大耳白兔，白胫骨取骨髓，采用直接贴壁培养法进行体外培养；倒置显微镜观察细胞形态，流式细胞术检测细胞表面抗原CIM4和CIM5的表达；以慢病毒为载体将外源基因增强型绿色荧光蛋白（EGFP）导入BMSCs内，观察其在细胞内持续表达的时间。结果分离培养的细胞在第5天可以观察到贴壁生长；培养10d融合可达80％，第1～5代细胞传代后增殖速度较快，平均倍增时间为48h，5代后细胞增殖速度明显减慢。流式细胞术鉴定发现BMSCs表面标志物为CD44（＋）和CIM5（-），慢病毒介导的EGFP基因转染的BMSCs2周后仍可产生绿色荧光。结论采用直接贴壁培养法进行体外培养可获得生长稳定、增殖能力强的BMSCs；慢病毒载体介导的EGFP基因转染的方法可将目的基因稳定地整合到细胞DNA中，并可在体外进行自我复制，转染成功后的BMSCs可在体外较长时间产生荧光，是一种可靠的标记方法。
Objective To establish an effective method of labeling rabbit bone mesenchymal stem cells in vitro. Methods Bone marrow was harvested from the tibias of 2-week old New Zealand white rabbits and cultured by the method of direct adherent culture. Biological characters of bone marrow mesenchyreal stem cells （BMSCs） were observed by an inverted microscope. CD44 and CD45 antigens of BMSCs were detected by flow cytometry. The lentiviruses were used to mediate the enhanced green fluorescent protein （EGFP） gene into the BMSCs. Results BMSCs could be observed 5 days after isolation. The primary ceils were confluent in single layer after being cultured for 10 days. The 1-5th generation BMSCs were noted to have great potential of proliferation, and the average time for cell doubling was 48 h. The muhiplicated ability of the BMSCs began to decrease since the 5th generation. Flow cytometry showed that the markers of BMSCs were CD44 ＋ , CD45-. BMSCs could express green fluorescence stablely after the transfection of EGFP gene by the lentivirus. Conclusion BMSCs cultured by direct adherent culture in vitro showed stable growth and rapid proliferation. Lentivirusmediated enhanced green fluorescent protein gene transfection could integrate EGFP gene into the BMSCs＇ DNA and express green fluorescence efficiently, and it is an effective labelling method of BMSCs.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2011年第12期2124-2126,共3页
Chinese Journal of Experimental Surgery
关键词
骨髓间充质干细胞
细胞培养
增强型绿色荧光蛋白
标记
Bone marrow mesenchymal stem cells
Cell culture
Enhanced green fluorescent protein
Label
