两种从骨髓中分离人间充质干细胞方法的比较研究

A comparative study of two methods for isolating human mesenchymal stem cells from bone marrow

目的比较F icoll(1.073 g/m l)密度梯度离心法分离人骨髓间充质干细胞(hum an bone m arrow derived m esen-chym al stem cells,hBMMSCs)与常用的Percoll分离法之间的差异。建立一种简便、实用的hBMMSCs分离方法。方法分别应用上述两种方法分离hBMMSCs,比较用两种方法从骨髓中分离的有核细胞得率及死亡率、贴壁细胞克隆数、细胞形态以及细胞表面标志。结果两种方法获得的有核细胞得率无显著差异(P>0.05);F icoll(1.073 g/m l)密度梯度离心法获得的有核细胞死亡率显著小于Percoll分离法(P<0.01);F icoll(1.073 g/m l)密度梯度离心法获得的hBMMSCs原代培养5 d的贴壁细胞克隆数/37.5 cm2显著多于Percoll分离法(P<0.05)。F icoll(1.073 g/m l)密度梯度离心法与Percoll分离法获得的hBMMSCs细胞形态均一,为纺锤形或三角形;F icoll(1.073 g/m l)密度梯度离心法与Percoll分离法获得的原代hBMMSCs CD105阳性表达率[(94.0±2.0)%vs(95.8±1.5)%]及CD34阴性表达率[(96.0±1.2)%vs(97±1.0)%]无显著差异(P>0.05)。结论与Percoll分离法相比较,F icoll(1.073 g/m l)密度梯度离心法分离hBMMSCs具有细胞死亡率较小,细胞得率较多的优点,是一种较好的hBMMSCs分离方法。

Objective To compare two density gradient centrifugation using Ficoll （ 1. 073 g/ml） and Percoil （1. 073 g/ml） as solutes respectively to separate human bone marrow derived mesenchymal stem cells （hBMMSCs） from bone marrow. Methods hBMMSCs were isolated from bone marrow of the iliac crest of voluntary normal adults by methods mentioned above respectively. Average number of nucleated cells and death rate after density gradient centrifugation with two solutes were compared and the hBMMSCs were observed in morphology and compared in cell shape, adherent cell clones, specific cell-markers after primary culture for 5 d. Results Average number of nucleated cells gained from two methods had no significant difference. Death rates of nucleated cells isolated by Ficoll （ 1. 073 g/ml） was markedly lower than that by Percoll （ 1. 073 g/ml） （P 〈0.01 ）. Number of adherent cell clones in 37.5 cm^2 culture flask after primary culture for 5 d in Ficoll （ 1. 073 g/ml） method was markedly higher than that of Percoll （ 1. 073 g/ml） method （P 〈0. 05）. hBMMSCs isolated by Ficoll （1. 073 g/ml） or Percoll （1. 073 g/ml） were both homogeneous spindle-like or triangle-like cells. Positive rates of CD105 and negative rates of CD34 in cells separated by Ficoll （ 1. 073 g/ml） or Pereoll （1. 073 g/ml） were similar （CD105：94.0±2.0% vs 95.8± 1.5% ; CD34： 96. 0 ± 1.2% vs 97 ± 1.0%）. Conclusion As compared to Percoll （ 1. 073 g/ml） method, density gradient centrifugation of Ficoll （ 1. 073 g/ml） has a lower cell death rate and higher number of adherent cell clones in 37.5 cm^2 culture flask after primary culture for 5 d. It is a good isolation method of human bone marrow derived mesenchymal stem cells.