摘要
目的体外建立MC3T3-E1细胞与RAW 264.7细胞间接共培养体系,观察AG490对共培养体系中破骨细胞形成的影响。方法 MC3T3-E1细胞经矿化诱导培养后,与RAW 264.7细胞进行间接共培养,经一定浓度AG490(1、5、10μmol/L)分别处理后,细胞计数试剂盒(CCK-8)检测细胞增殖,氮偶联法检测成骨细胞碱性磷酸酶(ALP)活性,实时荧光定量聚合酶链反应(PCR)检测成骨细胞相关基因骨保护素(OPG)、Ⅰ型胶原(COL1)、ALP、骨钙素(OCN)的表达,抗酒石酸酸性磷酸酶(TRAP)和苏木素染色检测破骨细胞的形成,蛋白印迹法(Western blot)检测破骨细胞中RANK的表达。采用单因素方差分析和双因素方差分析对数据进行统计分析。结果 AG490下调MC3T3-E1细胞矿化过程中OPG、COL1、ALP、OCN mRNA的表达(F_(OPG)=30.120,P_(OPG,AG490 1μmol/L)=0.021、P_(OPG,AG490 5μmol/L)=0.221、P_(OPG,AG490 10μmol/L)=0.003;F_(ALP)=67.352,P_(ALP,AG490 1μmol/L)=0.034、P_(ALP,AG490 5μmol/L)=0.001、P_(ALP,AG490 10μmol/L)=0.156;F_(COL1)=28.158,P_(COL1,AG490 1μmol/L)=0.054、P_(COL1,AG490 5μmol/L)=0.002、P_(COL1,AG490 10μmol/L)=0.4;F_(OCN)=125.375,P_(OCN,AG490 1μmol/L)<0.001、P_(OCN,AG490 5μmol/L)<0.001、P_(OCN,AG490 10μmol/L)<0.001)。与RAW264.7细胞单独培养作为对照组相比,MC3T3-E1细胞与RAW 264.7细胞间接共培养明显促进成熟破骨细胞的形成,AG490在这个过程中对破骨细胞的形成起到抑制作用(F=85.391,P_(共培养组-对照组)<0.001,P_(共培养+AG490组-共培养组)=0.035),同时与对照组相比,明显抑制破骨细胞中RANK蛋白的表达(F_(RANK)=376.25,P_(RANK,AG490 1μmol/L)=0.468、P_(RANK,AG490 5μmol/L)=0.003、P_(RANK,AG490 10μmol/L)<0.001)。结论 AG490抑制MC3T3-E1细胞成骨向分化,并通过影响OPG/RANKL/RANK信号轴的传递抑制成骨细胞与破骨前体细胞间接共培养过程中破骨细胞的形成。
Objective To establish a co-culture system of MC3T3-E1 cells and RAW 264.7 cells in vitro,and then explore the effect of AG490 on osteoclastogenesis in this system. Methods The mineralized MC3T3-E1 cells and RAW 264.7 cells were co-cultured after the addition of different concentrations of AG490（1μmol/L,5μmol/L,10μmol/L）. The cell proliferation was analyzed by CCK-8 assay,ALP activity was analyzed by a simultaneous-coupling azo dye method,and osteoblast-related genes OPG,COL1,ALP,OCN were analyzed by RT-PCR in mineralized MC3T3-E1 cells. TRAP and hematoxylin staining were performed to observe the osteoclastogenesis,and Western blot was adopted to analyze the RANK expression in osteoclasts. Data were evaluated by One-Way ANOVA and Two-Way ANOVA. Results AG490 inhibited the ALP activity and the expression of osteoblast-related genes OPG, COL1,ALP,OCN in mineralized MC3T3-E1 cells（FOPG=30.120,POPG,AG4901 μmol/L=0.021,POPG,AG4905 μmol/L=0.221,POPG,AG49010μmol/L=0.003;FALP=67.352,PALP,AG4901 μmol/L=0.034,PALP,AG4905 μmol/L=0.001,PALP,AG49010 μmol/L=0.156;FCOL1=28.158,PCOL1,AG4901 μmol/L=0.054,PCOL1,AG4905 μmol/L=0.002,PCOL1,AG49010 μmol/L=0.4;FOCN=125.375, POCN,AG4901 μmol/L〈0.001,POCN,AG4905 μmol/L〈0.001,POCN,AG49010 μmol/L〈0.001）. Compared with RAW264.7 cells alone group（control group）,the indirect co-culture of MC3T3-E1 cells and RAW264.7 cells successfully promoted osteoclast formation,which could be significantly inhibited by AG490（F=85.391,Pco-culture-control〈0.001,Pco-culture＋AG490-co-culture=0.035）,meanwhile the addition of AG490 down-regulated the RANK in the osteoclasts in protein level（FRANK=376.25,PRANK,AG4901μmol/L=0.468,PRANK,AG4905μmol/L=0.003,PRANK,AG49010μmol/L〈0.001）. Conclusions AG490 can inhibit osteoblastic differentiation in MC3T3-E1 cells,and might play an important role in osteoclast formation inhibition in co-culture of MC3T3-E1 cells and RAW 264.7 cells by blockade of OPG/RANKL/RANK signal axis.
出处
《中华口腔医学研究杂志（电子版）》
CAS
2017年第2期73-80,共8页
Chinese Journal of Stomatological Research(Electronic Edition)
基金
国家自然科学基金(81470718)
关键词
共同培养技术
核因子ΚB受体活化因子
AG490
成骨向分化
破骨向分化
Culture techniques
Receptor activator of nuclear factor-kappa B
Osteoblastic differentiation
Osteoclastic differentiation
