全反式维A酸对小鼠胚胎腭间充质细胞成骨分化的抑制作用及其机制

Inhibitory effect of all-trans retinoic acid on osteogenic differentiation of mouse embryonic palate mesenchymal cells and its possible mechanism

目的探讨全反式维A酸(atRA)对小鼠胚胎腭间充质细胞(MEPM)成骨分化的影响及其机制。方法分离培养MEPM细胞,在成骨诱导(OM)培养基中分别加入atRA 0.1和1.0μmol·L-1,分别于培养1,3,5,7和9 d用MTT法测定细胞存活率,化学比色法检测碱性磷酸酶(ALP)活性。培养21 d后,Von-Kossa染色观察矿化面积比。培养9 d后,逆转录PCR(RT-PCR)检测成骨相关基因Runx2、骨桥蛋白以及骨形态发生蛋白受体(Bmpr)1b,Bmpr2和Smad5 mRNA的表达水平。结果培养9 d后,与正常对照组相比,OM及OM+atRA 0.1和1.0μmol·L-1组细胞存活率明显降低(P<0.05);OM组细胞ALP活性最高,OM+atRA 1.0μmol·L-1组ALP活性明显低于OM组(P<0.05)。培养21 d后,Von-Kossa染色结果显示,OM+atRA 1μmol·L-1组矿化结节面积比为(3.56±1.24)%,明显低于OM组(10.33±2.29)%(P<0.05)。培养9 d后,OM组的Runx2相对表达各组内最高,OM+atRA 1.0μmol·L-1组与其相比约下调20%(P<0.05)。与正常对照组相比,OM组、OM+atRA 0.1和1.0μmol·L-1组骨桥蛋白mRNA表达明显升高(P<0.05)。OM组的Bmpr1b mRNA表达水平为正常对照组的1.6倍,OM+atRA 1.0μmol·L-1组的相对表达仅为OM组的33%(P<0.05)。OM+atRA 1.0μmol·L-1组Smad5 mRNA的表达水平明显低于OM组(P<0.05)。结论atRA能抑制MEPM的成骨分化,其作用机制可能与其下调Bmpr1b影响BMPR信号有关。

OBJECTIVE To investigate the effect and related mechanism of all-trans retinoic acid（ atRA）exposure on osteogenic differentiation of mouse embryonic palate masenchymal cells MEPM. METHODS MEPM were cultured in osteogenic medium（ OM） with atRA 0.1 and 1.0 μmol·L-1for 1,3,5,7 and 9 d.MTT assay was performed to measure the cell viability. The alkaline phosphatase（ ALP） activity was measured by chemical colorimetry. The cells were stained using the Von-Kossa technique to detect the formation of mineralization nodules after 21 d of culture. RT-PCR was performed to determine expression Runx2,osteopontin,bone morphogenetic protein receptor（ Bmpr） 1b,Bmpr2 and Smad5 mRNA. RESULTS The result of MTT on 9 d showed that,compared with normal control group,the cell viability of OM,OM＋atRA 0.1 and 1.0 μmol·L-1groups decreased significantly（ P 0.01）. Compared with normal control group,ALP activity of OM group increased significantly（ P〈0.05）,while the ALP activity of OM＋atRA0.1 and 1.0 μmol·L-1groups was lower than OM group（ P〈0.05）. On 21 d,the Von-Kossa staining results showed that the percentage of mineralization nodules formation of OM＋atRA 1.0 μmol·L-1group was（ 3.65±1.24） %,which was significantly lower than that of OM group（ 10.33±2.29） %（ P〈0.05）. On 9 d,the relative Run expression of OM group was the highest one in the four groups,while atRA 1.0 μmol·L-1treatment negatively regulated 20% in comparsion with OM group（ P〈0.05）. Compared with normal control group,the mRNA expression of osteopontin of OM,OM ＋ atRA 0. 1 and 1. 0 μmol·L-1groups increased significantly（ P 0.05）; BDNF mRNA expression of OM group was 2. 6-fold to normal control group,while that of OM＋atRA 1.0 μmol·L-1group was 33% to OM group（ P〈0.05）. The level of Smad5 mRNA of OM＋atRA 1.0 μmol·L-1group was significantly lower than that of OM group（ P〈0.05）. CONCLUSION atRA Might inhibit osteogenic differentiation of MEPM by down-regulated the expression of Bmpr1 b.