柚皮苷协同骨形态发生蛋白-2促进小鼠成骨细胞MC3T3-E1增殖和分化的研究

Effect of naringin combined with bone morphogenetic protein-2 on the proliferation and differentiation of MC3T3-E1 cells

目的研究柚皮苷(NAR)联合骨形态发生蛋白(BMP)-2对体外培养的成骨前体细胞MC3T3-E1增殖、碱性磷酸酶(ALP)活性及成骨相关基因ALP、骨钙素(OCN)、成骨特异性转录因子(Runx2)、Ⅰ型胶原(ColⅠ)表达的影响。方法以成骨细胞株MC3T3-E1为体外药效的试验模型,通过Alamar blue法检测第1、4和7天3种不同浓度NAR(10、100和1 000μmol·L-1)单独作用以及分别与50 ng·m L-1 BMP-2联合诱导对MC3T3-E1细胞增殖能力的影响。同时在第4天和7天测定成骨细胞内ALP活性,采用实时定量聚合酶链反应(q RT-PCR)检测成骨相关基因ALP、OCN、Runx2、ColⅠ的表达。结果单纯NAR刺激及联合BMP-2刺激能够促进细胞增殖,在NAR浓度为100μmol·L-1时达到高峰(P<0.05),且该浓度NAR与BMP-2联合作用时增殖效果大于两者单独作用(P<0.05)。单纯NAR刺激及联合BMP-2刺激4 d和7 d时均能促进细胞ALP活性,100μmol·L-1 NAR与BMP-2联合作用时ALP活性最强(P<0.05)。100~1 000μmol·L-1的NAR单独及联合作用在不同程度上能促进ALP、OCN、Runx2、ColⅠ成骨相关基因表达。结论 NAR能有效促进成骨细胞株MC3T3-E1增殖、分化,且适宜浓度的NAR和BMP-2有协同和促进作用。

Objective This study evaluates the biological effects of naringin （NAR） joint bone morphogenetic protein （BMP）-2 on the proliferation, alkaline phosphatase （ALP） activity, and expression of osteoblastogenic genes, such as Runt-related transcription factor 2 （Runx2）, collagen Ⅰ （ColⅠ）, ALP, and osteocalcin （OCN） of pre-osteoblasts. Methods Three different NAR concentrations （10, 100, and 1 000 μmol·L^-1） were applied, alone or combined with BMP-2（50 ng·mL^-1）, to restore the osteoblastogenesis of pre-osteoblasts （MC3T3-E1 cell line）. Cell numbers （proliferation） were evaluated at first, fourth, and seventh days by Alamar blue assay. ALP activity and the expression of osteoblastogenic genes, such as Runx2, ColⅠ, ALP, and OCN were analyzed by quantitative real-time polymerase chain reaction （qRT-PCR） at fourth and seventh day. Results Stimulation by NAR alone and in combination with BMP-2 for 1 day and 4 days could promote cell proliferation, which peaked at a concentration of 100 μmol·L^-1 NAR combined with BMP-2 could promote cell proliferation significantly （P〈0.05）. Stimulation by NAR alone and in combination with BMP-2 for 4 and 7 days could promote ALP activity and bone-related gene（ALP, OCN, Runx2, ColⅠ） expression. ALP expression was significantly promoted after stimulation of 100 μmol·L^-1 NAR and BMP-2 （P〈0.05）. Conclusion NAR exhibits promising potential for improving MC3T3-E1 proliferation and differentiation, and appropriate concentrations of NAR and BMP-2 show synergistic effect.