低氧预处理脐带间充质干细胞的旁分泌对成骨细胞功能的影响

Effect of paracrine of hypoxia preconditioned umbilical cord mesenchymal stem cells on function of osteoblasts

目的研究低氧预处理人脐带间充质干细胞(umbilical cord mesenchymal stem cells,UCMSC)旁分泌作用对人成骨细胞(MG-63)增殖、迁移及成骨的影响。方法分别于低氧及常氧条件下培养UCMSC,获取两种UCMSC条件培养基。实验分为3组:低氧培养基组、常氧培养基组、DMEM对照组,分别用3种不同的培养基常温培养MG-63。于培养1、3、5 d后用四氮唑盐(mosmann tetrazoline colorimetry,MTT)比色法检测细胞增殖情况,记录各组的光密度值进行统计学分析;用划痕法检测细胞在不同培养条件下的迁移能力;于培养21 d后进行茜素红染色检测各组成骨钙结节的形成。酶联免疫吸附法(ELISA)检测低氧及常氧条件培养基中血管内皮生长因子(vascular endothelial growth factor,VEGF)的含量。结果 MTT法检测结果表明,低氧及常氧培养基组的MG-63细胞在培养1、3、5 d后的增殖能力均大于DMEM对照组,差异有统计学意义(P<0.05),且低氧组大于常氧组,差异有统计学意义(P<0.05);划痕法结果为此两组细胞迁移能力大于DMEM对照组,且低氧组细胞迁移能力大于常氧组;钙结节染色结果显示低氧组成骨钙结节形成最多,常氧组次之,DMEM对照组最少。ELISA法检测低氧组VEGF含量高于常氧组,差异有统计学意义(P<0.01)。结论低氧预处理UCMSC可增强其旁分泌功能,促进成骨细胞MG-63的增殖、迁移及成骨。

Objective To study the paracrine effect of hypoxic preconditioned umbilical cord mesenchymal stem cells（ UCMSCs） on the proliferation,migration and osteogenic differentiation of osteoblasts. Methods UCMSCs were cultured under hypoxia and normal oxygen condition before two UCMSCs conditioned media were obtained. After that,MG-63 cells were cultured in three groups： hypoxia conditioned medium group,normoxia conditioned medium group and DMEM control group. The proliferation of MG-63 cells was detected by mosmann tetrazoline colorimetry（ MTT） method after 1,3 and 5days. The migratory ability of MG-63 cells was detected by scratch assay. After 21 days＇ culture,the formation of osteogenic calcium nodules was detected by Alizarin red staining. ELISA method was used to detect the content of vascular endothelial growth factor（ VEGF） in hypoxia and normoxia conditioned medium. Results The MTT test showed that the proliferation ability of MG-63 cells in hypoxia conditioned medium group and normoxia conditioned medium group was greater than in the DMEM control group. The difference was statistically significant（ P〈0. 05）. Furthermore,the proliferation ability of cells in hypoxia conditioned medium group were much greater than cells in normoxia culture medium group. The difference was statistically significant（ P〈0. 05）. Scrath assay showed that the migratory ability of MG-63 cells in hypoxia conditioned medium group and normoxia conditioned medium group was greater than cells in DMEM control group,and cells in hypoxia conditioned medium group was much greater than cells in normoxia conditioned medium group.After 21 days＇ culture,we found that the number of calcium nodules was the largest in hypoxia conditioned medium group,followed by normoxia conditioned medium group and DMEM control group. ELISA showed that the content of VEGF in hypoxia conditioned medium was higher than that in normoxia conditioned medium and the difference was statistically significant（ P〈0. 01）. Conclusion The paracrine function of UCMSCs can be enhanced by hypoxia,thus improving the proliferation,migration and osteogenic differentiation of osteoblasts.