不同成骨诱导体系对MC3T3-E1成骨分化的影响

Effects of Different Osteogenic Induction Systems on Osteogenic Differentiation of MC3T3-E1 Cells

目的:明确前成骨细胞MC3T3-E1最佳体外成骨诱导体系。方法:构建不同成骨诱导体系,分别为A组地塞米松0.1μmol/L、抗坏血酸50μg/mL及β-甘油磷酸钠10 mmol/L;B组地塞米松0.01μmol/L、抗坏血酸50μg/mL及β-甘油磷酸钠10 mmol/L;C组地塞米松1μmol/L、抗坏血酸50μg/mL及β-甘油磷酸钠10 mmol/L;D组抗坏血酸50μg/mL及β-甘油磷酸钠10 mmol/L;E组抗坏血酸100μg/mL及β-甘油磷酸钠5 mmol/L。对MC3T3-E1进行为期7 d、14 d的体外成骨诱导,通过碱性磷酸酶染色、茜素红染色对在不同成骨诱导体系作用下MC3T3-E1成骨分化的影响进行分析,通过RT-qPCR检测成骨分化相关基因ALP、Runx2、COL-1、OCN、OPN表达水平,比较不同成骨诱导体系的诱导性能。结果:B、D和E组茜素红定量最优,差异具有统计学意义(P<0.05),诱导7 d的RT-qPCR结果显示C组ALP、Runx2、COL-1的基因表达量最高(P<0.05)。诱导14 d的RT-qPCR结果显示:OCND组表达最高(P<0.05)。结论:地塞米松和抗坏血酸的浓度均对MC3T3-E1的成骨分化产生影响,C组1μmol/L地塞米松、50μg/mL抗坏血酸与10 mmol/Lβ-甘油磷酸钠早期成骨更佳,选择其作为早期成骨验证,D组50μg/mL抗坏血酸与10 mmol/Lβ-甘油磷酸钠晚期成骨更佳,但综合考虑,建议B组0.01μmol/L地塞米松、50μg/mL抗坏血酸与10 mmol/Lβ-甘油磷酸钠成骨诱导体系用于MC3T3-E1体外晚期成骨研究。

Objective:To determine the optimal osteogenic induction system in vitro for MC3T3-E1 osteoblasts.Methods:Different osteogenic induction systems were constructed,which were dexamethasone 0.1μmol/L,ascorbic acid 50μg/mL,andβ-sodium glycerophosphate 10 mmol/L in group A;dexamethasone 0.01μmol/L,ascorbic acid 50μg/mL,andβ-sodium glycerophosphate 10 mmol/L in group B;dexamethasone 1μmol/L,ascorbic acid 50μg/mL,andβ-sodium glycerophosphate 10 mmol/L in group C;ascorbic acid 50μg/mL andβ-sodium glycerophosphate 10 mmol/L in Group D;and ascorbic acid 100μg/mL andβ-sodium glycerophosphate 5 mmol/L in Group E.MC3T3-E1 cells were induced in vitro for 7 days and 14 days.The effects of alkaline phosphatase staining and alizarin red staining on osteogenic differentiation of MC3T3-E1 were analyzed.The expression levels of osteogenic differentiation related genes(ALP,Runx-2,COL-1,OCN,OPN)were detected by RT-qPCR.Results:There was a significant difference in the optimal quantification of alizarin among groups B,D,and E(P<0.05).The PCR results at 7 days after induction showed that the gene expression levels of ALP,Runx-2,and COL-1 in group C were the highest(P<0.05).PCR results at 14 days after induction showed that the expression of OCN in Group D was the highest(P<0.05).Conclusion:The concentrations of dexamethasone and ascorbic acid have an effect on the osteogenic differentiation of MC3T3-E1 cells.Group C was better for early osteogenesis and Group D was better for late osteogenesis.However,in comprehensive consideration,Group B is recommended for late osteogenesis.