目的 研究不同糖浓度对颌骨骨髓间充质干细胞（orofacial bonemesenchymal stem cells, OFMSCs）增殖和成骨分化的影响。方法 体外分离、培养OFMSCs，成骨、成脂、成软骨分化诱导及鉴定，并使用不同含糖量培养基（5.5、11、16.5、25、44 mmol/L）培养OFMSCs,以5.5 mmol/L基准糖浓度为对照组，其余组为实验组。采用CCK-8及流式细胞仪检测各组OFMSCs的增殖活性及增殖指数，OFMSCs成骨诱导后4、7 d检测碱性磷酸酶（ALP）活性，21 d进行茜素红染色及矿化定量分析，同时用RT-PCR检测3、7、14及21 d相关成骨基因Runx2、Osterix的表达。结果 培养的OFMSCs成骨诱导21 d后茜素红染色可见钙结节，成脂诱导14 d 后油红O染色可见红色脂滴，成软骨诱导14 d 后阿利新蓝染色可见蓝色胞浆；糖浓度在5.5～25 mmol/L促进OFMSCs的增殖，但随着糖浓度的继续增加（25～44 mmol/L），OFMSCs的增殖活性受抑制；成骨诱导时，随着培养液糖浓度升高，ALP活性呈剂量依赖性降低（P <0.05）；成骨诱导21 d ，茜素红染色和矿化定量分析显示，随着培养液糖浓度的升高，钙结节形成、矿化定量呈剂量依赖性减少；成骨诱导3、7、14及21 d ，RT-PCR检测结果显示，对照组Runx2、Osterix mRNA 表达量高于实验组（P <0.05）,各组Runx2、Osterix mRNA 表达量均出现先上调再下调的趋势。结论 在一定范围的糖浓度内，糖浓度升高可促进OFMSCs的增殖；而糖浓度升高对成骨分化呈抑制效应。
英文摘要:
Objective To determine the effects of different concentrations of glucose on the proliferation and osteogenic differentiation of orofacial bonemesenchymal stem cells (OFMSCs). Methods OFMSCs were primarily cultured and identified in vitro to undergo osteogenic/adipogenic/chondrogenic differentiation. The cells were exposed to osteogenic medium containing different levels of glucose: 5.5,11,16.5,25,44 mmol/L. The cell activity and proliferation index were measured using a cell counting kit (CCK)-8 and flow cytometry. The alkaline phosphatase (ALP) activity of the cells was measured at the 4th and 7th day. Alizarin red staining was carried out at the 21st day. RT-PCR detecting osteogenesis-related gene Runx2 and Osterix mRNA expression was performed at the 4th, 7th, 14th and 21st day. Results Osteogenesis induced calcium nodes was observed with Alizarin red staining at the 21st day. Adipogenic induced red lipid droplets was observed with Oil Red O staining at the 14th day. Chondrogenic induced blue cytoplasm was observed with Alcian blue staining at the 14th day. With 5.5 to 25 mmol/L glucose,OFMSCs proliferation was promoted.But when the concentration of glucose continued to increase (from 25 to 44 mmol/L),OFMSCs proliferation significantly reduced.The ALP activity decreased with glucose in a concentration-dependent manner (P <0.05). Osteogenesis induced Alizarin red staining and mineralization showed at the 21st day. The calcium nodes and mineralization quantity decreased with glucose in a concentration-dependent manner (P <0.05).The cells exposed to 5.5 mmol/L glucose had a higher level of expression of Runx2 and Osterix mRNA than the others (P <0.05).The experssion of Runx2 and Osterix mRNA in all groups showed a trend of rising first, followed by an obvious down regulation. Conclusion With certain limits,OFMSC proliferation is promoted by glucose. Osteogenic differentiation is inhibited by glucose in a concentration-dependent manner.
