骨髓间充质干细胞提取方法的改良

Improvement on the method of extracting bone marrow mesenchymal stem cells

背景:目前从大鼠骨髓中分离骨髓间充质干细胞仍以密度梯度离心法为主,虽可提取较纯的骨髓间充质干细胞,但步骤较繁琐,容易污染,且较昂贵。目的:试图寻找一种经济、方便及可靠的骨髓间充质干细胞提取方法。设计、时间及地点:以细胞为对象的对比观察实验,于2008-02/04在南方医科大学珠江医院中心实验室完成。材料:10只健康SD大鼠一侧前后肢提取出的骨髓间充质干细胞为实验组,另一侧前后肢提取出的骨髓间充质干细胞为对照组。方法:将实验组骨髓在含体积分数为0.10～0.15的胎牛血清、100IU/mL双抗及0.1%hepes液的DMEM培养基中培养,用注射器进行吹打,使骨髓与培养基充分混匀,吸取混合液,通过200目的筛网滤过后将其置于培养瓶中培养,提取较纯的骨髓间充质干细胞,对照组采用传统的密度梯度离心法提取骨髓间充质干细胞。主要观察指标:传代至第3代时,采用流式细胞仪鉴定提取的细胞,并从形态及数量上比较两种方法提取的细胞差异。结果:两种方法提取的骨髓间充质干细胞形态无明显差别,流式细胞仪检测通过改良法提取的细胞CD29+、CD44+、CD45-,可鉴定为骨髓间充质干细胞。两组间骨髓间充质干细胞数量无明显差异(P>0.05)。结论:实验采用的改良提取方法具有经济、操作简便等特点,是较为理想的大鼠骨髓间充质干细胞提取方法。

BACKGROUND： The current way of extracting bone marrow mesenchymal stem cells （BMSCs） from the rat is still density gradient centrifugation. BMSCs extracted by this way can be more pure, but it is more cumbersome steps, easily contaminated, and more expensive. OBJECTIVE： To find a method to extract BMSCs from the rat, which is more economic, convenient and reliable. DESIGN, TIME AND SETTING： The control cell experiment was performed at the Central Laboratory of Zhujiang Hospital of Southern Medical University from February to April 2008. MATERIALS： Ten Sprague Dawley rats were used to harvest BMSCs. BMSCs from one side served as an experimental group and BMSCs from another side served as a control group. METHODS： BMSCs in the experimental group were incubated in DMEM supplemented with 0.10-0.15 volume fraction of fetal bovine serum, 100 IU/mL double antibody and 0.1% hepes solution, and then hit with a syringe, misce bene. The mixture was filtered with a sieve of 200 mesh/cm^2 and incubated in a culture flask. Pure BMSCs were isolated. BMSCs were harvested by density gradient centrifugation in the control group. MAIN OUTCOME MEASURES： At the third passage, BMSCs were identified by flow cytometry. Cell difference in morphous and quantity was compared. RESULTS： No significant difference in morphous of BMSCs was detected between both groups. Cell CD29^＋, CD44^＋, CD45^- were identified by flow cytometry, which could be defined as BMSCs. No significant difference in quantity of BMSCs was detected between both groups （P 〉 0.05）. CONCLUSION： This is an improved method to extract BMSCs primarily, which is more economical, easier to operate.