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目的探讨二甲双胍对高糖介导的成骨细胞增殖受限、凋亡及功能蛋白表达损害的干预作用。方法体外培养原代小鼠颅骨成骨细胞,分为正常糖浓度(5.5 mmol·L~(-1)葡萄糖)组,高糖(25 mmol·L~(-1)葡萄糖)组,二甲双胍低、中、高浓度(25 mmol·L~(-1)葡萄糖+25、50、100μmol·L~(-1)二甲双胍)组,分别干预48、72 h,MTT法测定细胞增殖活性,流式细胞仪检测细胞凋亡率。各实验组细胞加入成骨诱导培养基,干预时间延长至1、2、3 wk,比色法和放射免疫法检测碱性磷酸酶(ALP)、骨钙素的分泌水平。结果在相同干预时限,与正常糖浓度组比较,高糖组细胞增殖能力降低、早期凋亡率增加(均P<0.05);二甲双胍低、中、高浓度组,随二甲双胍浓度增高,细胞增殖能力呈上升趋势(P<0.05),而细胞早期凋亡率呈下降趋势(P<0.05)。干预时间由48 h延长至72 h,各组细胞增殖能力和早期凋亡率呈不同程度增加。干预时间延长至1、2、3 wk;正常糖浓度组细胞ALP和骨钙素的分泌水平呈增加趋势(P<0.05),而高糖组则呈下降趋势(P<0.05)。在相同干预时限,二甲双胍干预组细胞ALP和骨钙素分泌水平随二甲双胍浓度增高而增加(P<0.05)。结论二甲双胍能够拮抗成骨细胞的糖毒性损害,保护成骨细胞的成骨能力,且在一定的浓度范围内呈剂量依赖性。
Objective To investigate the effects of metformin on the hyperglycemia-mediated osteoblast proliferation, apoptosis and the impaired expression of functional proteins. Methods Primary cultured mouse osteoblasts were cultured in vitro and were divided into normal glucose concentration (5.5 mmol·L -1) glucose group, high glucose (25 mmol·L -1 glucose) group, metformin low, Medium and high concentration (25 mmol·L -1 glucose + 25, 50 and 100 μmol·L -1 metformin) for 48 and 72 h respectively. MTT assay was used to determine the cell proliferation. Flow cytometry Detect the rate of apoptosis. The osteoblast-inducing medium was added into each experimental group, and the intervention time was prolonged to 1, 2 and 3 weeks. The levels of alkaline phosphatase (ALP) and osteocalcin were detected by colorimetry and radioimmunoassay. Results Compared with the normal glucose concentration group, the proliferation of high glucose group was decreased and the early apoptosis rate was increased (all P <0.05) at the same time of intervention. In the low, medium and high concentrations of metformin group, with the increase of metformin concentration, the cell proliferation ability (P <0.05), while the early apoptotic rate showed a decreasing trend (P <0.05). Intervention time from 48 h to 72 h, the proliferation of cells in each group and early apoptosis rate increased to varying degrees. The intervention time was prolonged to 1, 2, and 3 weeks. The levels of ALP and osteocalcin increased in normal glucose group (P <0.05), but decreased in high glucose group (P <0.05). Metformin intervention group cells ALP and osteocalcin levels increased with metformin concentration increased (P <0.05) at the same intervention time. Conclusion Metformin can antagonize the osteotoxicity of osteoblasts and protect the osteogenic capacity of osteoblasts in a dose-dependent manner.