二甲基乙二酰基甘氨酸对小鼠胚胎成骨细胞前体细胞增殖和分化的影响

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

目的 :探讨低氧模拟剂二甲基乙二酰基甘氨酸(dimethyloxalylglycine,DMOG)对小鼠胚胎成骨细胞前体细胞(MC3T3-E1)的增殖、成骨分化及血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响。方法:将MC3T3-E1细胞接种到培养板24h后,实验组培养基中分别加入50μM(50μM组)和200μM(200μM组)的DMOG,对照组加入完全培养液。分别于培养1、3、5d时采用MTT法检测MC3T3-E1细胞增殖情况,5、10d行碱性磷酸酶(alkaline phosphatase,ALP)染色及ALP活性检测成骨细胞分化,21d用茜素红染色检测MC3T3-E1细胞钙结节的形成并进行定量分析,采用ELISA法检测MC3T3-E1培养3d情况时上清液中的VEGF蛋白含量,并用实时荧光定量PCR法检测MC3T3-E1细胞VEGF mRNA的相对表达量。结果:培养1d时对照组、50μM组和200μM组的MC3T3-E1的光密度(optical density,OD)值分别为0.041±0.009、0.074±0.019、0.086±0.044,3d时分别为0.123±0.027、0.148±0.020、0.224±0.061,5d时分别为0.297±0.044、0.325±0.084、0.354±0.038,1d、3d时50μM组和200μM组与同时间点对照组比较均有显著性差异(P<0.05),3d时50μM组与200μM组有显著性差异(P<0.05)。培养5d和10d时对照组ALP染色颜色较深,50μM组颜色中等,200μM组颜色较浅;5d时对照组ALP活性为1.943±0.072,50μM组为1.632±0.051,200μM组为1.319±0.065;10d时对照组ALP活性为3.734±0.067,50μM组为3.381±0.070,200μM组为2.831±0.086。三组间同时间点比较均有统计学差异(P<0.05),同组10d时与5d时比较均有统计学差异(P<0.05)。茜素红染色200μM组可见少量红色结节,50μM组可见中等量红色结节,对照组可见大量红色结节;对照组茜素红含量(μg/ml)为56.178±7.940,50μM组为41.922±2.438,200μM组为31.929±1.922,三组间比较均有统计学差异(P<0.05)。培养3d时对照组细胞培养上清液中VEGF蛋白含量(ng/孔)为9.063±0.603,50μM组为12.123±0.870,200μM组为15.540±0.581,三组间比较均有统计学差异(P<0.05);50μM组VEGF mRNA表达量为对照组的1.792±0.067,200μM组为对照组的3.963±0.092,三组间比较均有统计学差异(P<0.05)。结论 :低氧模拟剂DMOG可促进MC3T3-E1细胞增殖和VEGF表达,抑制其成骨分化。

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.