摘要
目的研究野黄芩苷的体外促成骨分化作用。方法利用荧光素酶报告基因系统检测野黄芩苷对稳定转染bmp2启动子的MC3T3-bmp2-Luc细胞中bmp2转录活性的影响;体外培养小鼠颅骨来源的前体成骨细胞系MC3T3-E1和小鼠多能间充质干细胞样成纤维细胞C3H10T1/2,采用MTT的方法检测野黄芩苷对细胞增殖的影响;p-NPP法检测野黄芩苷对成骨细胞内碱性磷酸酶活性的影响以及通过茜素红染色的方法考察野黄芩苷对钙化结节形成的影响;Real-time PCR进一步验证野黄芩苷长时间诱导后对成骨细胞bmp2 m RNA水平的影响。结果野黄芩苷可以在20μmol/L显著上调bmp2转录活性（P〈0.001）;在1~20μmol/L的剂量浓度范围内,野黄芩苷作用72 h后对MC3T3-E1细胞增殖不明显但也无杀伤作用。野黄芩苷在两种实验用细胞中均能剂量依赖地上调ALP的活性,其中10μmol/L和20μmol/L最为显著（P〈0.05）,并且在C3H10T1/2细胞中该作用呈时间依赖性。同时也观察到24 d诱导后,野黄芩苷（5~10μmol/L）可以增加成骨细胞钙化结节的数目,且诱导12 d后,在5~20μmol/L浓度下上调bmp2 m RNA的水平。结论野黄芩苷具有体外促成骨分化的活性,有潜力成为抗骨质疏松候选化合物。
Objective To study the effects of scutellarin on osteoblast differentiation in vitro. Methods Cell model MC3T3-bmp2-Luc stably transfected with mouse bmp2 promoter-luciferase reporter vector was chosen to test the expression of the mouse bmp2 by scutellarin treatment. Then MC3T3-E1 cells and C3H10T1/2 cells were cultured in vitro to evaluate the effects of scutellarin on osteoblast differentiation. The osteogenic differentiation capacity was evaluated by 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide（MTT） assay, alkaline phosphatase（ALP） assay（p-NPP methods）, alizarin red staining and real-time PCR. Results Scutellarin was effective in up-regulating the transcription activity of bmp2 in the dose of 20 μmol/L（P 〈0.001）. The MTT assay showed that it neither promoted the proliferation nor induced cell death in the dose ranging between 1- 20 μmol/L in MC3T3-E1 cells. Scutellarin could increase ALP activity in both MC3T3-E1 and C3H10T1/2 cell lines, especially in the dose of 10 and 20 μmol/L（P〈0.05）. In the dose ranging between 5- 10 μmol/L, the mineralization of C3H10T1/2 cells was enhanced by treatment with scutellarin. The m RNA synthesis of bmp2 was also promoted by scutellarin treatment for 12 days. Conclusion Scutellarin shows the effects on osteoblast differentiation, deserving further investigation as a potential anabolic agent.
出处
《中国医药生物技术》
2016年第1期27-31,共5页
Chinese Medicinal Biotechnology
基金
国家自然科学基金海外及港澳学者合作研究基金(81328024)
关键词
骨形态发生蛋白质2
碱性磷酸酶
成骨分化
野黄芩苷
Bone morphogenetic protein 2
Alkaline phosphatase
Osteogenic differentiation
Scutellarin
