两种分离脐血间充质干细胞的方法比较

Comparison of two isolation methods for umbilical cord blood mesenchymal stem cells

背景:目前分离脐血间充质干细胞的方法很多,尚没有确定一种为最有效的方法。目的:寻找一种最为可靠的脐血间充质干细胞分离方法。方法:应用Percoll分离液法和羟乙基淀粉沉降法对脐血进行分离得到单核细胞,在含体积分数15%新生牛血清的DMEM/F12培养基中进行培养并传代。观察不同分离方法脐血单核细胞的回收率,每次传代的时间和细胞增值速度,培养过程中间充质干细胞形态的变化情况,并用流式细胞仪检测第3代细胞表面标志物CD90、CD44、CD34的表达。结果与结论:与Percoll分离液法相比羟乙基淀粉沉降法获得的单核细胞多,单核细胞回收率高(P<0.01),第1次传代时间短(P<0.01)。然而两种方法获得的细胞经培养在形态的变化和表面标志物CD90、CD44、CD34的表达上差异并无显著性意义(P>0.05)。所以羟乙基淀粉沉降法的分离效率较高,培养时间短,但是并不能获得质量较高的脐血间充质干细胞。

BACKGROUND： There is no standard method to isolate umbilical cord blood mesenchymal stem cells （MSCs）.OBJECTIVE： To discuss an optimized isolation method of human umbilical cord blood MSCs.METHODS： Monocytes were isolated from umbilical cord blood using the Percoll separation medium method and hydroxyethyl starch sedimentation respectively.Cells were cultured in DMEM/F12 containing 15% newborn calf serum.The main outcome indictors were recovery rate of monocytes,generation time,cell proliferation,morphological change,and the surface markers expressed on umbilical cord blood MSCs measured by flow cytometer.RESULTS AND CONCLUSION： Compared with the Percoll separation medium method,the recovery rate of monocytes increased and the first passage time reduced using the hydroxyethyl starch sedimentation （P 0.01）.But the morphological change and surface markers CD90,CD44,CD34 expression of two methods have no differences （P 0.05）.Therefore,the hydroxyethyl starch sedimentation has higher isolation efficiency and the shorter culture time than Percoll separation medium method.However,the quality of umbilical cord blood MSCs which are isolated by two methods has no difference.