大鼠骨髓间充质干细胞定向成骨细胞分化中碱性磷酸酶的变化

目的　研究大鼠骨髓间充质干细胞药物诱导定向成骨细胞分化特性及分化过程中碱性磷酸酶的变化。方法　用质量浓度为 1.0 73kg·L-1的Percoll分离骨髓间充质干细胞 ,再连续进行 3次换液以纯化骨髓间充质干细胞。加药物 β 甘油磷酸钠、地塞米松和维生素C诱导骨髓间充质干细胞定向成骨细胞分化。在加药后 5、10、15、2 0d用比色法检测细胞中碱性磷酸酶含量。改良Gomori染色 ,统计阳性细胞百分率。结果　梯度密度离心和黏附贴壁法联合使用可得到均一的骨髓间充质干细胞。用比色法测得在第 5、10、15、2 0d ,碱性磷酸酶含量 (A40 5)分别为 0 .391± 0 .0 2 14、0 .4 345± 0 .0 2 31、0 .8781± 0 .0 2 96和 0 .933± 0 .0 2 36 ;对照组为 0 .16 5 6± 0 .0 2 4 2、0 .15 2 8± 0 .0 2 15、0 .14 5 3± 0 .0 2 0 4和 0 .14 35± 0 .0 2 16。同时期改良Gomori染色阳性细胞率分别为 10 .35 %、11.4 8%、35 .2 8%和 4 6 .7%。结论　大鼠骨髓间充质干细胞经药物诱导后可以分化成成骨细胞。在分化过程中碱性磷酸酶含量从给药后的 10d左右才开始显著升高。在此过程中改良Gomori染色结果与其保持一致

Change of ALPase during the differentiation into osteoblast from rat marrow mesenchymal stem cells

Objective To study the potential of rat marrow mesenchymal stem cells differentiating into osteoblast through drug induction and the change of ALPase during the differentiation. Methods The rat marrow stem cells were isolated by Percoll (1.073 kg·L -1) and purified by continuously changing medium. The third passage cells had been induced by dexamethasome, β-glycerophosphate and ascorbic acid-2-phosphate for 20 days. On 5 d,10 d, 15 d and 20 d after induction, the content of ALPase was analyzed by color match and stained with modified Gomori. The percentage of positive cells was also calculated. Results Homogeneous mesenchymal stem cells were separated by gradient density centrifugation and adherence. On 5 d ,10 d,15 d and 20 d after induction, the A 405 of ALPase was 0.391±0.0214, 0.4345±0.0231, 0.8781±0.0296 and 0.933±0.0236, respectively, while that of the controls was 0.1656±0.0242, 0.1528±0.0215, 0.1453±0.0204, and 0.1435±0.0216. The percentage of modified Gomori stained positive cells was 10.35%, 11.48%, 35.28% and 46.7% on 5 d, 10 d, 15 d and 20 d, respectively. Conclusion Combination of the agents can differentiate mesenchymal stem cells into osteoblast. The content of ALPase in osteoblast rises from the 10th day to the 15th day.