CAJ | 학술논문

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。
목적：탐토전반식유 A 산(atRA)대소서배태악간충질세포(MEPM)성골분화적영향급기궤제。방법분리배양 MEPM 세포，재성골유도(OM)배양기중분별가입 atRA 0.1화1.0μmol??L－1，분별우배양1，3，5，7화9 d 용 MTT 법측정세포존활솔，화학비색법검측감성린산매( ALP)활성。배양21 d 후，Von￣Kossa염색관찰광화면적비。배양9 d 후，역전록 PCR(RT￣PCR)검측성골상관기인 Runx2、골교단백이급골형태발생단백수체(Bmpr)1b， Bmpr2화 Smad5 mRNA 적표체수평。결과배양9 d 후，여정상대조조상비，OM 급 OM＋atRA 0.1화1.0μmol??L－1조세포존활솔명현강저(P<0.05)；OM 조세포 ALP 활성최고，OM＋atRA 1.0μmol??L－1조 ALP 활성명현저우 OM 조(P<0.05)。배양21 d 후，Von￣Kossa 염색결과현시，OM＋atRA 1μmol??L－1조광화결절면적비위(3.56±1.24)％，명현저우 OM 조(10.33±2.29)％( P <0.05)。배양9 d 후，OM 조적 Runx2상대표체각조내최고，OM＋atRA 1.0μmol??L－1조여기상비약하조20％(P<0.05)。여정상대조조상비，OM 조、OM＋atRA 0.1화1.0μmol??L－1조골교단백 mRNA 표체명현승고(P<0.05)。 OM 조적 Bmpr1b mRNA 표체수평위정상대조조적1.6배，OM＋atRA 1.0μmol??L－1조적상대표체부위OM 조적33％(P<0.05)。 OM＋atRA 1.0μmol??L－1조 Smad5 mRNA 적표체수평명현저우 OM 조(P<0.05)。결론atRA 능억제MEPM 적성골분화，기작용궤제가능여기하조 Bmpr1b 영향 BMPR 신호유관。
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-1 for 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-1 groups 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+atRA 0.1 and 1.0 μmol??L-1 groups was lower than OM group(P<0.05). On 21 d, the Von￣Kossa stai￣ning results showed that the percentage of mineralization nodules formation of OM+atRA 1.0 μmol??L-1 group 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 at￣RA 1.0 μmol??L-1 treatment 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-1 groups 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-1 group was 33% to OM group(P<0.05) . The level of Smad5 mRNA of OM+atRA 1.0 μmol??L-1 group 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 Bmpr1b.