骨形态发生蛋白2诱导C2C12和MC3T3-E1的成骨分化与自噬

摘要

BACKGROUND:A series of studies indicate that autophagy is closely linked with differentiation. Bone morphogenetic protein 2 (BMP-2) is the classical pathway for C2C12 and MC3T3-E1 osteoblast differentiation, and the ideal model to study osteogenic differentiation process. OBJECTIVE:To observe the relationship between autophagy and BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation. METHODS:Real-Time PCR was applied to detect osteogenic differentiation and autophagy related index after C2C12 and MC3T3-E1 were induced with BMP-2 (100μg/L) for 72 hours. The osteogenic index alkaline phosphatase in BMP-2-induced MC3T3-E1 and C2C12 cultured with different concentrations of 3-methyladenine (0, 1, 5, 10 mmol/L) was determined with alkaline phosphatase staining. Western blot analysis was applied to detect LC3-I/II expression levels in C2C12 and MC3T3-E1 induced with BMP-2 for different time points (0, 12, 24, 48, 72, 96 hours). RESULTS AND CONCLUSION:The autophagy-related mRNA and protein expression showed an increasing tendency and autophagy-related protein LC3 levels was increased, which was associated with the time, during the BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation. Meanwhile, alkaline phosphatase expression levels were inhibited by autophagy in the process of osteogenic differentiation. Therefore, there is a close relationship between autophagy and the BMP-2-induced celllines C2C12, MC3T3-E1 osteoblast differentiation.%背景：一系列研究表明自噬与分化有密切联系；骨形态发生蛋白2是诱导C2C12、MC3T3-E1成骨分化经典途径，是研究成骨分化过程的理想模型。　　目的：观察自噬与骨形态发生蛋白2诱导细胞株C2C12、MC3T3-E1成骨分化的关系。　　方法：Real-Time PCR检测MC3T3-E1与C2C12在骨形态发生蛋白2(100μg/L)诱导培养3 d后成骨与自噬相关指标变化。碱性磷酸酶染色检测不同浓度3-甲基腺嘌呤(0，1，5，10 mmol/L)对骨形态发生蛋白2(100μg/L)诱导培养7 d MC3T3-E1与C2C12成骨指标碱性磷酸酶变化，Western Blot检测C2C12和MC3T3-E1在骨形态发生蛋白2(100μg/L)诱导不同时间点(0，12，24，48，72，96 h)LC3-Ⅰ/Ⅱ蛋白表达水平。　　结果与结论：在骨形态发生蛋白2诱导细胞株 C2C12、MC3T3-E1成骨分化过程中，诱导自噬相关 mRNA与蛋白水平均有增高趋势，且自噬相关蛋白LC3水平增高与时间相关。同时，抑制自噬成骨分化过程中碱性磷酸酶表达水平降低。因此，自噬与骨形态发生蛋白2诱导细胞株C2C12、MC3T3-E1成骨分化过程有密切关系。