二甲双胍对成牙骨质细胞成骨作用的研究

Study on the effect of metformin on osteogenesis of cementoblasts

目的:研究二甲双胍(metformin,MF)对成牙骨质细胞(cementoblasts,CB)增殖、分化、矿化能力的影响,为临床牙骨质缺损、牙根修复提供新的用药思路。方法:对成牙骨质细胞系OCCM-30分别给予不同浓度的MF培养,MTS法检测CB的增殖;实时荧光定量PCR法(quantitative real-time polymerase chain reaction,qPCR)及蛋白免疫印迹法检测骨涎蛋白(bone sialoprotein,BSP)和Runt相关转录因子2(runt-related transcription factor 2,Runx 2)在基因及蛋白层面的表达;碱性磷酸酶(alkaline ph osph atase,ALP)活性测定及茜素红染色法检测MF对CB矿化的影响。结果:不同浓度的MF作用于OCCM-30细胞24、48、72小时后,200μmol/L和400μmol/L MF组对细胞的增殖能力具有促进作用(P<0.05),高浓度1600μmol/L MF组在作用细胞72小时后对细胞增殖产生抑制作用(P<0.05)。与对照组相比,用200μmol/L MF处理OCCM-30细胞12、24、48小时后,Runx 2和BSP mRNA及蛋白的表达量明显增高(P<0.05)。200μmol/L MF作用OCCM-30细胞3天时ALP活性高于对照组(P<0.05),作用7天时则无统计学差异(P>0.05)。200μmol/L MF作用OCCM-30细胞21天后,矿化结节形成百分率明显高于对照组(P<0.01)。结论:一定浓度范围内的MF能促进CB的增殖、分化及矿化,提高CB的成骨能力。

Objective:To study the effects of metformin(MF)on the proliferation,differentiation and mineralization ability of cementoblasts(CB),and to Pprovide new medicine ideas for clinical cementum defect and root restoration.Methods:Cementoblast cell line OCCM-30 cells were cultured with different concentrations of MF and the proliferation of CB was detected by MTT assay.Quantitative real-time polymerase chain reaction(qPCR)and Western blotting were used to detect the expression of bone sialoprotein(BSP)and runt-related transcription factor 2(Runx2)at the gene and protein levels.The effect of MF on CB mineralization was determined by alkaline phosphatase(ALP)activity and alizarin red staining.Results:After treatment of OCCM-30 cells with different concentrations of MF for 24,48 and 72h,200μmol/L and 400μmol/L MF groups promoted cell proliferation(P<0.05),while high concentration of 1600μmol/L MF group inhibited cell proliferation after treatment for 72h(P<0.05).Compared with the control group,the mRNA and protein expressions of Runx2 and BSP in OCCM-30 cells,were significantly increased in OCCM-30 cells treated with 200μmol/L MF for 12,24 and 48 h(P<0.05).The ALP activity of OCCM-30 cells treated with 200μmol/L MF for 3 days was higher than that of control group(P<0.05),and there was no statistical difference at 7 days(P>0.05).After treated with 200μmol/L MF for 21 days,the percentage of mineralized nodules formed in OCCM-30 cells was significantly higher than that in control group(P<0.01).Conclusion:MF in a certain concentration range can promote the proliferation,differentiation and mineralization of CB,and improve the osteogenic ability of CB.