人骨髓间充质干细胞体外分离培养、鉴定及标记

Isolation culture,identification and labeling of human mesenchymal stem cells in vitro

目的探讨人骨髓间充质干细胞(hMSCs)体外分离培养、鉴定和标记的方法以及生物学特性。方法无菌条件下穿刺人髂后上嵴抽取骨髓,采用直接贴壁法分离纯化hMSCs,体外扩增,用免疫细胞化学法及流式细胞仪分别对培养的hMSCs进行鉴定,同时检测第3代和同代溴脱氧尿嘧啶(5bromo2’deoxyuridine,BrdU)标记的hMSCs细胞周期。结果体外培养的原代hMSCs在培养24小时内可见少量贴壁细胞,7天达到融合,免疫细胞化学示CD44和CD105表达阳性,CD34表达阴性,流式细胞仪示CD44阳性率为89.64%,CD34为0.11%。大部分hMSCs核抗BrdU染色阳性,阳性率达90%。细胞周期显示第3代和同代BrdU标记的hMSCs约有90%的细胞处于G0/G1期,第10代为80%。结论hMSCs在体外很容易分离培养和扩增,但随着传代次数的增加,hMSCs变得宽大扁平且增殖速度减慢,出现衰老现象;用BrdU标记hMSCs对其细胞周期无明显影响,可作为标记细胞的一种有效手段。

Objective To investigate the methods of isolation,culture,identification and labeling of human mesenchymal stem cells (hMSCs) and their biological characters. Methods Puncture posterior superior iliac spine of healthy adults under sterile condition. hMSCs were isolated and purified using direct anchoring methods and identified using immunocytochemical methods and flow cytometry. Cell cycles of the third,tenth passage and BrdU-labeled hMSCs were tested by flow cytometry. Results Primary cultured hMSCs adhered to plastic surface within 24 hours and reached 80% confluence within 7 days. Immunocytochemistry showed hMSCs are positive for CD44 and CD105 and negative for CD34. BrdU stains nucleus of some hMSCs. The positive rate of CD44 is 89.64% while CD34 is 0.11% by flow cytometry. Cell cycles showed 90% cells of the third passage and BrdU-labeled hMSCs are at G0/G1 phase,80% at the tenth passage. Conclusion hMSCs can be isolated,cultured and expanded easily. With the increased numbers of subcultivation,hMSCs become widen and flatten and the proliferation rate steps down. BrdU-label has little effect on the cell cycle of hMSCs,so it can be used as an effective method of labeling cells.