机械压应力下骨硬化蛋白对成牙骨质细胞功能影响及机制的体外研究
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摘要
目的探究骨硬化蛋白(SOST)对处于机械压应力中的永生化成牙骨质细胞(OCCM-30)的功能影响及其相关的机制。方法用不同浓度SOST培养液(0、25、50、100 ng·mL-1)处理细胞后依靠四点弯曲细胞力学加载器对细胞加载大小为2 000μstrain、频率是0.5 Hz的单轴压应力6 h,用免疫印迹法检测β-连环蛋白(β-catenin)、磷酸化的细胞信号转导分子p-smad1/5/8、细胞信号转导分子smad1/5/8的蛋白水平;用碱性磷酸酶(ALP)活性检测法检测ALP活性;用荧光实时定量PCR检测核心结合蛋白因子2(Runx-2)、骨钙素(OCN)、骨涎蛋白(BSP)以及细胞核因子κB受体活化因子(RANKL)、骨保护因子(OPG)的表达。结果 p-smad1/5/8随着SOST浓度增加,呈减低的趋势,而β-catenin、smad1/5/8未有显著差异性。在仅对细胞加力时ALP活性降低,随SOST浓度升高,ALP活性逐渐发生下降,Runx-2、OCN和BSP的表达也都呈现降低趋势,RANKL的表达随着SOST增加而升高,OPG则随之下降。结论压应力下,SOST的升高会对骨形态发生蛋白(BMP)/smad通路产生抑制作用,对β-catenin表达未产生明显改变。外源性SOST对于BMP存在反馈性的负向调节作用。压应力下SOST对OCCM-30的矿化功能是抑制的,其机制或许是一方面利用BMP信号通路对成骨相关因子Runx2、OCN、BSP等实现下调,另一方面提高了与破牙骨质有关分子RANKL/OPG的比率。
Objective The purpose of this study is to investigate the potential effects of sclerostin(SOST) on the biological funtions and re lated me chanisms of cementoblasts under mechanical stress. Methods OCCM-30 cells were treated with varying doses of SOST(0, 25, 50, and 100 ng·mL-1) and were loaded with uniaxial compressive stress(2 000 μ strain with a frequency of 0.5 Hz) for six hours. Western blot was utilized to detect the expressions of β-catenin, p-smad1/5/8, and smad1/5/8 proteins. Alkaline phosphatase(ALP) activity was determined, and reverse transcription polymerase chain reaction was used to measure the expressions of runt-related transcription factor 2(Runx-2), osteocalcin(OCN), bone sialoproteins(BSP), receptor activator of NF-κB ligand(RANKL) and osteoprotegerin(OPG) mRNA. Results The expression of p-smad1/5/8 was significantly downregulated with increasing SOST.β-catenin and smad1/5/8 exhibited no difference. ALP activity decreased under me chanical compressive stress with increasing SOST con cen trations. Runx-2 expression was reduced with increasing SOST concentrations, and a similar trend was observed for the BSP and OCN expressions. When the SOST concentration was enhanced, RANKL expression gradually increased, whereas the expression of OPG decreased. Conclusion Under mechanical comprehensive stress, SOST can adjust the bone morpho genetic protein(BMP)/smad signal pathway. Osteosclerosis inhibits the mineralization of cementoblasts under mechanical compressive stress, which may be achieved by inhibiting the expressions of osteogenesis factors(Runx2, OCN, BSP, and others) and by promoting the ratio of cementoclast-related factors(RANKL/OPG) through BMP signal pathways.
Objective The purpose of this study is to investigate the potential effects of sclerostin(SOST) on the biological funtions and re lated me chanisms of cementoblasts under mechanical stress. Methods OCCM-30 cells were treated with varying doses of SOST(0, 25, 50, and 100 ng·mL-1) and were loaded with uniaxial compressive stress(2 000 μ strain with a frequency of 0.5 Hz) for six hours. Western blot was utilized to detect the expressions of β-catenin, p-smad1/5/8, and smad1/5/8 proteins. Alkaline phosphatase(ALP) activity was determined, and reverse transcription polymerase chain reaction was used to measure the expressions of runt-related transcription factor 2(Runx-2), osteocalcin(OCN), bone sialoproteins(BSP), receptor activator of NF-κB ligand(RANKL) and osteoprotegerin(OPG) mRNA. Results The expression of p-smad1/5/8 was significantly downregulated with increasing SOST.β-catenin and smad1/5/8 exhibited no difference. ALP activity decreased under me chanical compressive stress with increasing SOST con cen trations. Runx-2 expression was reduced with increasing SOST concentrations, and a similar trend was observed for the BSP and OCN expressions. When the SOST concentration was enhanced, RANKL expression gradually increased, whereas the expression of OPG decreased. Conclusion Under mechanical comprehensive stress, SOST can adjust the bone morpho genetic protein(BMP)/smad signal pathway. Osteosclerosis inhibits the mineralization of cementoblasts under mechanical compressive stress, which may be achieved by inhibiting the expressions of osteogenesis factors(Runx2, OCN, BSP, and others) and by promoting the ratio of cementoclast-related factors(RANKL/OPG) through BMP signal pathways.
引文
[1]Ramirez-Echave JI, Buschang PH, Carrillo R, et al. Histologic evaluation of root response to intrusion in mandibular teeth in beagle dogs[J]. Am J Orthod Dentofacial Orthop,2011, 139(1):60-69.
[2]吴卫锋.成牙骨质细胞的研究进展[J].国际口腔医学杂志, 2011, 38(1):95-97.Wu WF. Research progress on cementoblast[J]. Int J Stomatol, 2011, 38(1):95-97.
[3]包丽娜.机械应力刺激对成牙骨质细胞BSP/OPN mRNA表达影响的体外研究[D].成都:四川大学, 2007.Bao LN. Effects of mechanical stress stimulation on the expressionofBSP/OPNmRNAincementoblasts:anin vitro study[D]. Chengdu:Sichuan University, 2007.
[4]林婷婷,陆尔奕.骨硬化蛋白在骨组织改建中作用的分子机制[J].国际口腔医学杂志, 2012, 39(2):269-272.Lin TT, Lu EY. Molecular mechanism of sclerostin on bone remoldling[J]. Int J Stomatol, 2012, 39(2):269-272.
[5]Kamiya N, Kobayashi T, Mochida Y, et al. Wnt inhibitors Dkk1 and Sost are downstream targets of BMP signaling through the type IA receptor(BMPRIA)in osteoblasts[J]. J Bone Miner Res, 2010, 25(2):200-210.
[6]Papanicolaou SE, Phipps RJ, Fyhrie DP, et al. Modulation of sclerostin expression by mechanical loading and bone morphogenetic proteins in osteogenic cells[J]. Biorheology,2009, 46(5):389-399.
[7]Lin C, Jiang X, Dai Z, et al. Sclerostin mediates bone response to mechanical unloading through antagonizing Wnt/betacatenin signaling[J]. J Bone Miner Res, 2009, 24(10):1651-1661.
[8]Bao X, Liu Y, Han G, et al. The effect on proliferation and differentiation of cementoblast by using sclerostin as inhibitor[J]. Int J Mol Sci, 2013, 14(10):21140-21152.
[9]Huang L, Meng Y, Ren A, et al. Response of cementoblastlike cells to mechanical tensile or compressive stress at physiologicallevelsinvitro[J].MolBiolRep,2009,36(7):1741-1748.
[10]Nemoto E, Koshikawa Y, Kanaya S, et al. Wnt signaling inhibits cementoblast differentiation and promotes proliferation[J]. Bone, 2009, 44(5):805-812.
[11]Kim TH, Lee JY, Baek JA, et al. Constitutive stabilization ofβ-catenin in the dental mesenchyme leads to excessive dentin and cementum formation[J]. Biochem Biophys Res Commun, 2011, 412(4):549-555.
[12]Zhao M, Xiao G, Berry JE, et al. Bone morphogenetic protein2 induces dental follicle cells to differentiate toward a cementoblast/osteoblast phenotype[J]. J Bone Miner Res, 2002,17(8):1441-1451.
[13]ZhaoM,BerryJE,SomermanMJ.Bonemorphogenetic protein-2 inhibits differentiation and mineralization of cementoblasts in vitro[J]. J Dent Res, 2003, 82(1):23-27.
[14]李书琴.骨形成蛋白2调控成牙骨质细胞中sclerostin表达变化的初步研究[D].重庆:重庆医科大学, 2015.Li SQ. The preliminary research on expression of sclerostin mediated by BMP2 in OCCM30[D]. Chongqing:Chongqing Medical University, 2015.
[15]Polson A, Caton J, Polson AP, et al. Periodontal response after tooth movement into intrabony defects[J]. J Periodontol,1984, 55(4):197-202.
[16]D’Errico JA, MacNeil RL, Takata T, et al. Expression of bone associated markers by tooth root lining cells, in situ and in vitro[J]. Bone, 1997, 20(2):117-126.
[17]Liu W, Toyosawa S, Furuichi T, et al. Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures[J]. J Cell Biol, 2001, 155(1):157-166.
[18]Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype[J]. Endocr Rev,1993, 14(4):424-442.
[19]Karsenty G. Role of Cbfa1 in osteoblast differentiation and function[J]. Semin Cell Dev Biol, 2000, 11(5):343-346.
[20]Hunter GK, Goldberg HA. Modulation of crystal formation by bone phosphoproteins:role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein[J].Biochem J, 1994, 302(Pt 1):175-179.
[21]Hunter GK, Kyle CL, Goldberg HA. Modulation of crystal formation by bone phosphoproteins:structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation[J]. Biochem J, 1994, 300(Pt 3):723-728.
[22]Matias MA, Li H, Young WG, et al. Immunohistochemical localisation of extracellular matrix proteins in the periodontium during cementogenesis in the rat molar[J]. Arch Oral Biol, 2003, 48(10):709-716.
[23]黄兰.机械压应力调控成牙骨质细胞生物学行为的体外研究[D].成都:四川大学, 2010.Huang L. The study on the biological behavior of cementoblast in the regulation of mechanical compressive stress in vitro[D]. Chengdu:Sichuan University, 2010.
[24]Boabaid F, Berry JE, Koh AJ, et al. The role of parathyroid hormone-related protein in the regulation of osteoclastogenesis by cementoblasts[J]. J Periodontol, 2004, 75(9):1247-1254.
[25]Kostenuik PJ, Shalhoub V. Osteoprotegerin:a physiological and pharmacological inhibitor of bone resorption[J]. Curr Pharm Des, 2001, 7(8):613-635.
[2]吴卫锋.成牙骨质细胞的研究进展[J].国际口腔医学杂志, 2011, 38(1):95-97.Wu WF. Research progress on cementoblast[J]. Int J Stomatol, 2011, 38(1):95-97.
[3]包丽娜.机械应力刺激对成牙骨质细胞BSP/OPN mRNA表达影响的体外研究[D].成都:四川大学, 2007.Bao LN. Effects of mechanical stress stimulation on the expressionofBSP/OPNmRNAincementoblasts:anin vitro study[D]. Chengdu:Sichuan University, 2007.
[4]林婷婷,陆尔奕.骨硬化蛋白在骨组织改建中作用的分子机制[J].国际口腔医学杂志, 2012, 39(2):269-272.Lin TT, Lu EY. Molecular mechanism of sclerostin on bone remoldling[J]. Int J Stomatol, 2012, 39(2):269-272.
[5]Kamiya N, Kobayashi T, Mochida Y, et al. Wnt inhibitors Dkk1 and Sost are downstream targets of BMP signaling through the type IA receptor(BMPRIA)in osteoblasts[J]. J Bone Miner Res, 2010, 25(2):200-210.
[6]Papanicolaou SE, Phipps RJ, Fyhrie DP, et al. Modulation of sclerostin expression by mechanical loading and bone morphogenetic proteins in osteogenic cells[J]. Biorheology,2009, 46(5):389-399.
[7]Lin C, Jiang X, Dai Z, et al. Sclerostin mediates bone response to mechanical unloading through antagonizing Wnt/betacatenin signaling[J]. J Bone Miner Res, 2009, 24(10):1651-1661.
[8]Bao X, Liu Y, Han G, et al. The effect on proliferation and differentiation of cementoblast by using sclerostin as inhibitor[J]. Int J Mol Sci, 2013, 14(10):21140-21152.
[9]Huang L, Meng Y, Ren A, et al. Response of cementoblastlike cells to mechanical tensile or compressive stress at physiologicallevelsinvitro[J].MolBiolRep,2009,36(7):1741-1748.
[10]Nemoto E, Koshikawa Y, Kanaya S, et al. Wnt signaling inhibits cementoblast differentiation and promotes proliferation[J]. Bone, 2009, 44(5):805-812.
[11]Kim TH, Lee JY, Baek JA, et al. Constitutive stabilization ofβ-catenin in the dental mesenchyme leads to excessive dentin and cementum formation[J]. Biochem Biophys Res Commun, 2011, 412(4):549-555.
[12]Zhao M, Xiao G, Berry JE, et al. Bone morphogenetic protein2 induces dental follicle cells to differentiate toward a cementoblast/osteoblast phenotype[J]. J Bone Miner Res, 2002,17(8):1441-1451.
[13]ZhaoM,BerryJE,SomermanMJ.Bonemorphogenetic protein-2 inhibits differentiation and mineralization of cementoblasts in vitro[J]. J Dent Res, 2003, 82(1):23-27.
[14]李书琴.骨形成蛋白2调控成牙骨质细胞中sclerostin表达变化的初步研究[D].重庆:重庆医科大学, 2015.Li SQ. The preliminary research on expression of sclerostin mediated by BMP2 in OCCM30[D]. Chongqing:Chongqing Medical University, 2015.
[15]Polson A, Caton J, Polson AP, et al. Periodontal response after tooth movement into intrabony defects[J]. J Periodontol,1984, 55(4):197-202.
[16]D’Errico JA, MacNeil RL, Takata T, et al. Expression of bone associated markers by tooth root lining cells, in situ and in vitro[J]. Bone, 1997, 20(2):117-126.
[17]Liu W, Toyosawa S, Furuichi T, et al. Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures[J]. J Cell Biol, 2001, 155(1):157-166.
[18]Stein GS, Lian JB. Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype[J]. Endocr Rev,1993, 14(4):424-442.
[19]Karsenty G. Role of Cbfa1 in osteoblast differentiation and function[J]. Semin Cell Dev Biol, 2000, 11(5):343-346.
[20]Hunter GK, Goldberg HA. Modulation of crystal formation by bone phosphoproteins:role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein[J].Biochem J, 1994, 302(Pt 1):175-179.
[21]Hunter GK, Kyle CL, Goldberg HA. Modulation of crystal formation by bone phosphoproteins:structural specificity of the osteopontin-mediated inhibition of hydroxyapatite formation[J]. Biochem J, 1994, 300(Pt 3):723-728.
[22]Matias MA, Li H, Young WG, et al. Immunohistochemical localisation of extracellular matrix proteins in the periodontium during cementogenesis in the rat molar[J]. Arch Oral Biol, 2003, 48(10):709-716.
[23]黄兰.机械压应力调控成牙骨质细胞生物学行为的体外研究[D].成都:四川大学, 2010.Huang L. The study on the biological behavior of cementoblast in the regulation of mechanical compressive stress in vitro[D]. Chengdu:Sichuan University, 2010.
[24]Boabaid F, Berry JE, Koh AJ, et al. The role of parathyroid hormone-related protein in the regulation of osteoclastogenesis by cementoblasts[J]. J Periodontol, 2004, 75(9):1247-1254.
[25]Kostenuik PJ, Shalhoub V. Osteoprotegerin:a physiological and pharmacological inhibitor of bone resorption[J]. Curr Pharm Des, 2001, 7(8):613-635.