Effects of dimethyloxalylglycine on proliferation and osteogenic differentiation in MC3T3-E1 cells

English Keywords:Dimethyloxalylglycine  MC3T3-E1 cells  Alkaline phosphatase  Vascular endothelial growth factor  Osteogenic differentiation

English Abstract:

  【Abstract】 Objectives: To explore the effects of hypoxia mimic agent dimethyloxalylglycine(DMOG) on the proliferation, osteogenic differentiation and vascular endothelial growth factor(VEGF) expression of MC3T3-E1 cell. Methods: After MC3T3-E1 cells were seeded on plate for 24 hours, DMOG was added to the culture medium with concentrations of 50μM(50μM group) and 200μM(200μM group) respectively, while in the control group no DMOG was added. The proliferation of MC3T3-E1 cells was detected by MTT assay at the 1st, 3rd and 5th day of cells culture. The osteoblast differentiation was determined by alkaline phosphatase activity analysis and ALP staining at the 5th and 10th day. The formation of calcium nodules in MC3T3-E1 cells was detected by alizarin red staining and quantitative analysis at the 21st day. The level of VEGF in the supernatant of MC3T3-E1 cells was detected by ELISA method, and the gene expression of VEGF was also detected by real-time fluorescence quantitative PCR. Results: At the 1st day of cells culture, the values of light density (optical density, OD) in the control group, the 50μM group and the 200μM group were 0.041±0.009, 0.074 ±0.019, and 0.086±0.044 respectively. At the 3rd day, the values of OD in the control group, the 50μM group, and the 200μM group were 0.123±0.027, 0.148±0.020, 0.224±0.061 respectively. At the 5th day, the values of OD in the control group, the 50μM group and the 200μM group were 0.297±0.044, 0.325±0.084, 0.354±0.038 respectively. At the 1st day and the 3rd day, the DMOG results of the 50μM and 200μM group were significantly different from that of the control group(P<0.05). At the 3rd day, the DMOG result of 50μM group was significantly different from that of 200μM group. ALP staining showed that the color was deeper in the control group, moderate in the 50μM group, and shallow in the 200μM group. At the 5th day, the alkaline phosphatase activity was 1.943 ±0.072 in the control group, 1.632±0.051 in the 50μM group, 1.319±0.065 in the 200μM group. At the 10th day, the alkaline phosphatase activity was 3.734±0.067 in the control group, 3.381±0.070 in the 50μM group, 2.831±0.086 in the 200μM group. Alkaline phosphatase activity was significantly different between each two groups(P<0.05). Alizarin red staining and quantitative analysis showed a small amount of red nodules in the 200μM group, a moderate amount red nodules in the 50μM group, while a large number of red nodules in the control group. The alizarin red content was 56.178 ±7.940 in the control group, 41.922±2.438 in the 50μM group, 31.929±1.922 in the 200μM group. There was significant difference of alizarin red content(μg/ml) between each two groups(P<0.05). The VEGF content in the supernatant was 9.063 ±0.603 in the control group, 12.123±0.870 in the 50μM group, 15.540±0.581 in the 200μM group. There was significant difference of VEGF content between each two groups(P<0.05). The VEGF gene expression level was 1.792±0.067 in the 50μM group, 3.963±0.092 in the 200μM group, and there was significant difference of VEGF gene expression level between each two groups(P<0.05). Conclusions: Under normal oxygen condition, hypoxia mimicking agent DMOG significantly promotes proliferation and VEGF expression, while inhibites osteogenic differentiation in MC3T3-E1 cell.