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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、44mmol/L)培养OFMSCs,以5.5mmol/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、7d检测碱性磷酸酶(ALP)活性,21d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21d相关成骨基因Runx2、Osterix的表达。结果培养的OFMSCs成骨诱导21d后茜素红染色可见钙结节,成脂诱导14d后油红O染色可见红色脂滴,成软骨诱导14d后阿利新蓝染色可见蓝色胞浆;糖浓度在5.5~25mmol/L促进OFMSCs的增殖,但随着糖浓度的继续增加(25~44mmol/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21d,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21d,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。
Objective To investigate the effects of different concentrations of glucose on the proliferation and osteogenic differentiation of OFMSCs. Methods OFMSCs were isolated and cultured in vitro. Osteogenesis, adipogenesis and chondrogenic differentiation were induced and identified. OFMSCs were cultured in different glucose medium (5.5, 11, 16.5, 25 and 44 mmol / L) Sugar concentration of the control group, the remaining group for the experimental group. The proliferation and proliferation index of OFMSCs in each group were detected by CCK-8 and flow cytometry. The activities of alkaline phosphatase (ALP) of OFMSCs were detected 4 and 7 days after osteogenic induction on OFMSCs. Alizarin red staining and mineralization quantitative analysis were performed on day 21, At the same time, the expression of Runx2 and Osterix in 3, 7, 14 and 21d osteoblasts were detected by RT-PCR. RESULTS OFMSCs were cultured for 21 days. Alizarin red staining showed calcium nodules. After adipogenic differentiation for 14 days, red lipid droplets were observed in oil red O staining, and blue cytoplasm was observed after alginate blue staining for 14 days. 5.5 ~ 25mmol / L could promote the proliferation of OFMSCs. However, with the increasing of glucose concentration (25 ~ 44mmol / L), the proliferation activity of OFMSCs was inhibited. At the time of osteogenic induction, (P <0.05). After osteogenic induction for 21 days, alizarin red staining and mineralization quantitative analysis showed that with the increase of glucose concentration in the culture medium, the formation and mineralization of calcium nodules decreased in a dose-dependent manner. At 3, 7, 14 and 21 days after osteogenic induction, the expression of Runx2 and Osterix mRNA in the control group was higher than that in the experimental group (P <0.05) by RT-PCR. The expressions of Runx2 and Osterix mRNA in all groups increased first and then decreased the trend of. Conclusion Within a certain range of sugar concentration, the increase of sugar concentration can promote the proliferation of OFMSCs; while the increase of sugar concentration has an inhibitory effect on osteogenic differentiation.