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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 investigate the effects of hypoxic preconditioning on proliferation, migration and osteogenesis of human osteoblast (MG-63) induced by paracrine effect of umbilical cord mesenchymal stem cells (UCMSC). Methods UCMSCs were cultured under hypoxic and normoxic conditions, and two kinds of UCMSC conditioned media were obtained. The experiment was divided into three groups: hypoxia medium group, normoxia medium group and DMEM control group. MG-63 was cultured at room temperature in three kinds of different media respectively. The cell proliferation was detected by MTT assay after 1, 3, and 5 days of culture. The optical density of each group was recorded and statistically analyzed. The cell viability Under the conditions of migration ability; After 21 days of culture, alizarin red staining was performed to detect the formation of osteocalcin. The levels of vascular endothelial growth factor (VEGF) in hypoxia and normoxia conditioned medium were detected by enzyme-linked immunosorbent assay (ELISA). Results The results of MTT assay showed that the proliferation of MG-63 cells cultured in hypoxia and normoxia medium for 1, 3 and 5 days were higher than that of DMEM control group (P <0.05), and The hypoxia group was larger than normoxia group, the difference was statistically significant (P <0.05). Scratching results showed that the migrating ability of the two groups was greater than DMEM control group, The results of staining showed that the formation of osteocalcin was the most in hypoxia group, followed by normoxia group and the least in DMEM control group. The level of VEGF in hypoxia group was higher than that in normoxia group by ELISA (P <0.01). Conclusion Hypoxic preconditioning of UCMSC can enhance its paracrine function and promote the proliferation, migration and osteogenesis of osteoblasts MG-63.