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

doi:10.7518/hxkq.2019.02.007

华西口腔医学杂志 ›› 2019, Vol. 37 ›› Issue (2): 162-167.doi: 10.7518/hxkq.2019.02.007

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

柏思羽,陈悦,戴红卫,黄兰()

重庆医科大学附属口腔医院正畸科口腔疾病与生物医学重庆市重点实验室重庆市高校市级口腔生物医学工程重点实验室,重庆 401147

收稿日期:2018-08-20 修回日期:2019-01-20 出版日期:2019-04-01 发布日期:2019-04-28
通讯作者: 黄兰 E-mail:lanhuang29@126.com
作者简介:柏思羽,硕士,E-mail： 382761236@qq.com
基金资助:
国家自然科学青年基金(81300914);2016年重庆高校创新团队建设计划(CXTDG201602006)

Effect of sclerostin on the functions and related mechanisms of cementoblasts under mechanical stress

Siyu Bai,Yue Chen,Hongwei Dai,Lan Huang()

Dept. of Orthodontics, Stomatological Hospital of Chongqing Medical University, Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing 401147, China

Received:2018-08-20 Revised:2019-01-20 Online:2019-04-01 Published:2019-04-28
Contact: Lan Huang E-mail:lanhuang29@126.com
Supported by:
The National Natural Science Foundation of China for Young Scholars(81300914);Program for Innovation Team Building at Institutions of Higher Education in Chongqing in 2016(CXTDG201602006)

摘要/Abstract

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2 000 μstrain、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。

关键词: 成牙骨质细胞, 机械压应力, 成骨矿化, 骨硬化蛋白, 信号通路

Abstract:

Objective The purpose of this study is to investigate the potential effects of sclerostin (SOST) on the biological funtions and related mechanisms 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-smad 1/5/8 was significantly downregulated with increasing SOST. β-catenin and smad1/5/8 exhibited no difference. ALP activity decreased under mechanical compressive stress with increasing SOST concentrations. 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 morphogenetic 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.

Key words: cementoblast, mechanical stress, mineralization, sclerostin, signal pathway

中图分类号:

Q257

柏思羽,陈悦,戴红卫,黄兰. 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[J]. 华西口腔医学杂志, 2019, 37(2): 162-167.

Siyu Bai,Yue Chen,Hongwei Dai,Lan Huang. Effect of sclerostin on the functions and related mechanisms of cementoblasts under mechanical stress[J]. West China Journal of Stomatology, 2019, 37(2): 162-167.

图/表 5

表1

图1

图2

图3

图4

参考文献 25

[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. doi: 10.1016/j.ajodo.2009.07.014 URL
[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 expression of BSP/OPN mRNA in cementoblasts: an in 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. doi: 10.1359/jbmr.090806 URL
[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/betaca-tenin signaling[J]. J Bone Miner Res, 2009,24(10):1651-1661. doi: 10.1359/jbmr.090411 URL
[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. doi: 10.3390/ijms141021140 URL
[9]	Huang L, Meng Y, Ren A , et al. Response of cementoblast-like cells to mechanical tensile or compressive stress at physiological levels in vitro[J]. Mol Biol Rep, 2009,36(7):1741-1748. doi: 10.1007/s11033-008-9376-3 URL
[10]	Nemoto E, Koshikawa Y, Kanaya S , et al. Wnt signaling inhibits cementoblast differentiation and promotes proliferation[J]. Bone, 2009,44(5):805-812. doi: 10.1016/j.bone.2008.12.029 URL
[11]	Kim TH, Lee JY, Baek JA , et al. Constitutive stabilization of β-catenin in the dental mesenchyme leads to excessive dentin and cementum formation[J]. Biochem Biophys Res Commun, 2011,412(4):549-555. doi: 10.1016/j.bbrc.2011.07.116 URL
[12]	Zhao M, Xiao G, Berry JE , et al. Bone morphogenetic protein 2 induces dental follicle cells to differentiate toward a cementoblast/osteoblast phenotype[J]. J Bone Miner Res, 2002,17(8):1441-1451. doi: 10.1359/jbmr.2002.17.8.1441 URL
[13]	Zhao M, Berry JE, Somerman MJ . Bone morphogenetic protein-2 inhibits differentiation and mineralization of cementoblasts in vitro[J]. J Dent Res, 2003,82(1):23-27. doi: 10.1177/154405910308200106 URL
[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. doi: 10.1902/jop.1984.55.4.197 URL
[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. doi: 10.1016/S8756-3282(96)00348-1 URL
[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. doi: 10.1083/jcb.200105052 URL
[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. doi: 10.1210/edrv-14-4-424 URL
[19]	Karsenty G . Role of Cbfa1 in osteoblast differentiation and function[J]. Semin Cell Dev Biol, 2000,11(5):343-346. doi: 10.1006/scdb.2000.0188 URL
[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. doi: 10.1042/bj3020175 URL
[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. doi: 10.1042/bj3000723 URL
[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. doi: 10.1016/S0003-9969(03)00131-6 URL
[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. doi: 10.1902/jop.2004.75.9.1247 URL
[25]	Kostenuik PJ, Shalhoub V . Osteoprotegerin: a physiological and pharmacological inhibitor of bone resorption[J]. Curr Pharm Des, 2001,7(8):613-635. doi: 10.2174/1381612013397807 URL

目标	引物（5’—3’）	片段大
基因	引物（5’—3’）	小/bp
SOST	上游：CAAGGCAGTGTTTCCACCTT	168
	下游：CACGTAGCCCAACATCACAC
Runx-2	上游：GGACGAGGCAAGAGTTTCA	123
	下游：CTGTCTGTGCCTTCTTGGTT
OCN	上游：CTGACCTCACAGATGCCAAG	98
	下游：AAGGTAGTGAACAGACTCCG
BSP	上游：CTGAAGAAAAACGGGGTCTTTAAG	136
	下游：ATTGGAGACGGCGATAGTTC
GAPDH	上游：CCTCAAGATTGTCAGCAAT	141
	下游：CCATCCACAGTCTTCTGGGT
RANKL	上游：GCCTCCCGCTCCATGTTC	101
	下游：TTAGGATCCATCTGCGCTC
OPG	上游：TCAGAAAGGAAATGCAACACA	205
	下游：CCGTTTTATCCTCTCTACACT

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

Effect of sclerostin on the functions and related mechanisms of cementoblasts under mechanical stress

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 25

相关文章 15

编辑推荐

Metrics

本文评价

[1]	晚晓芳, 何海燕, 吕佳岭, 伍宇婕, 钟冠男, 徐晓梅. 周期性张应力作用下Hippo-YAP信号通路调控人牙周膜细胞自噬[J]. 华西口腔医学杂志, 2023, 41(3): 260-268.
[2]	邬浩, 李莹, 王玉琢, 于继泽, 包幸福, 胡敏. 重组人成纤维细胞生长因子21调节成牙骨质细胞矿化作用及其机制研究[J]. 华西口腔医学杂志, 2023, 41(2): 140-148.
[3]	陆佳艺, 伍倩琪, 陈伊燕, 叶蕾蕾, 苏媛. 牙龈卟啉单胞菌来源的脂多糖通过X盒结合蛋白1调控脂肪细胞胰岛素信号通路的机制研究[J]. 华西口腔医学杂志, 2022, 40(2): 148-154.
[4]	王林, 王熙, 季楠, 李海梅, 蔡世新. 机械激活性离子通道压电蛋白Piezo1通过Notch信号通路介导牙周膜干细胞成骨分化作用机制研究[J]. 华西口腔医学杂志, 2020, 38(6): 628-636.
[5]	翁旭, 李劲松, 范松. 长链非编码RNA PCGEM1通过转化生长因子β2/Smad2信号通路调控口腔鳞状细胞癌侵袭和转移的机制研究[J]. 华西口腔医学杂志, 2020, 38(5): 550-557.
[6]	惠婷, 张广灿, 冯丹丹, 汲平. 神经肽P物质及骨形态发生蛋白信号通路在ST2细胞成骨分化过程中的作用[J]. 华西口腔医学杂志, 2018, 36(4): 378-383.
[7]	马玉, 李淑慧, 丁欣欣, 吴佩玲. 肿瘤坏死因子-α对牙周膜干细胞骨向分化及Notch信号通路的影响[J]. 华西口腔医学杂志, 2018, 36(2): 184-189.
[8]	董晓菲, 王会, 蓝菁. 蓬乱蛋白2在高脂血症大鼠种植体周围早期表达的实验研究[J]. 华西口腔医学杂志, 2018, 36(1): 82-86.
[9]	陈一文,高尚,许彤彤,张佳慧,李金城,李金成,张慧彦,卢金金,胡敏,刘志辉. 大鼠正畸牙移动过程中牙周膜骨硬化蛋白的表达[J]. 华西口腔医学杂志, 2016, 34(3): 239-243.
[10]	陈悦,李书琴,黄兰,戴红卫. 骨形态发生蛋白2对成牙骨质细胞中硬化蛋白表达调控机理的研究[J]. 华西口腔医学杂志, 2016, 34(3): 244-247.
[11]	王飞,张慧宇,窦予昕,李适廷,张纲,谭颖徽. 降钙素基因相关肽通过Hippo通路调控小鼠骨髓间充质干细胞成骨分化的实验研究[J]. 华西口腔医学杂志, 2016, 34(3): 286-290.
[12]	刘鑫黄兴徐智云杨德琴. 视黄酸信号通路调控咽弓神经嵴影响斑马鱼牙齿发育的研究[J]. 华西口腔医学杂志, 2016, 34(2): 115-120.
[13]	许来青刘媛媛罗晶晶姜曚郭文豪郑广宁. 成牙骨质细胞瘤的临床及影像表现分析[J]. 华西口腔医学杂志, 2015, 33(4): 419-422.
[14]	李书琴杨珊任嫒姝戴红卫. 张应力刺激下Wnt/β-catenin信号通路对成牙骨质细胞Runx2表达调控的体外研究[J]. 华西口腔医学杂志, 2015, 33(1): 35-39.
[15]	尚针针李欣孙惠强贾可丽. MC3T3-E1细胞流体剪切力敏感信号通路的基因芯片研究[J]. 华西口腔医学杂志, 2014, 32(5): 509-512.