低强度高频率振动对成骨细胞生物学特性的影响

doi:10.13418/j.issn.1001-165x.2018.02.010

摘要/Abstract

摘要：

目的　研究低强度高频率振动（low-magnitude high-frequency vibration, LMHFV）对成骨细胞生物学特性的影响。方法　建立LMHFV加载MC3T3-E1细胞模型，观察不同频率LMHFV对MC3T3-E1细胞OPG/RANKL浓度比的影响，获得OPG/RANKL浓度比最高的频率（F）为后续研究频率；以0 Hz为对照，观察LMHFV对MC3T3-E1细胞碱性磷酸酶 (ALP)、骨钙素(OCN) mRNA和蛋白活性，及钙化结节形成的影响；LMHFV加载形成的条件培养液（CMF）孵育RAW264.7细胞，观察CMF对破骨细胞抗酒石酸酸性磷酸酶（TRAP）染色、多核破骨细胞形成、TRAP mRNA及蛋白活性的影响；观察LMHFV对MC3T3-E1细胞环氧化酶2(COX-2)蛋白水平的表达及COX-2抑制剂NS-398对LMHFV影响MC3T3-E1细胞分化的作用。结果　30 Hz LMHFV获得OPG/RANKL浓度比最高，促进ALP、OCN mRNA及蛋白活性增加，增加钙化结节形成。30 Hz LMHFV形成的CM抑制RAW264.7细胞向多核破骨细胞分化，抑制TRAP mRNA及活性；LMHFV可诱导COX-2蛋白水平增加，NS-398能抑制LMHFV促进成骨细胞分化。结论　30 Hz的LMHFV对MC3T3-E1细胞OPG/RANKL浓度比及成骨分化具有积极的影响，通过调控成骨细胞OPG/RANKL浓度比间接抑制骨吸收，COX-2通路参与了LMHFV对成骨细胞生物学特性的调节作用。

关键词: 骨质疏松, 　低强度高频率振动, 　成骨细胞, 　生物学特性

Abstract:

Objective　To study the effects of low-magnitude high-frequency vibration (LMHFV) on the biological characteristics of osteoblasts.　Methods　To establish a model of MC3T3-E1 cells loaded with LMHFV, and observe the effects of LMHFV at different frequencies on the concentration ratio of OPG / RANKL in MC3T3-E1 cells, the frequency with the highest OPG / RANKL concentration ratio (F) was the frequency of follow-up studies. With 0 Hz as a control, to observe the effects of LMHFV on the expression of akialine phosphatase (ALP), osteocalcin (OCN) mRNA and protein activity, and calcified nodules in MC3T3-E1 cells; RAW264.7 cells were incubated with conditioned medium (CMF) loaded with LMHFV, and to observe the effect of CMF on TRAP staining, multinucleated osteoclast formation, TRAP mRNA and protein activity in osteoclasts; to study the expression of cyclooxygenase-2 (COX-2) protein level in MC3T3-E1 cells loaded with LMHFV and the effect of COX-2 inhibitor NS-398 on the differentiation of MC3T3-E1 cells induced by LMHFV.　Results　The highest concentration ratio of OPG / RANKL was obtained at 30 Hz in LMHFV, which promoted the increase of ALP, OCN mRNA and protein activity and increased the formation of calcified nodules. CM formed by LMHFV at 30 Hz inhibited the differentiation of RAW264.7 cells into multinucleated osteoclasts and inhibited the TRAP mRNA and protein activity. LMHFV induced an increase in COX-2 protein level and NS-398 inhibited LMHFV-induced osteoblast differentiation.　Conclusion　LMHFV at 30 Hz had a positive effect on OPG / RANKL concentration ratio and osteogenic differentiation in MC3T3-E1 cells, indirectly suppresses bone resorption by regulating the osteoblast OPG / RANKL concentration ratio, the COX-2 pathway is involved in the regulation of osteoblast biological properties by LMHFV.

Key words: Osteoporosis; , Low-intensity high-frequency vibration, Osteoblast; , Biological characteristics

查丁胜,邓轩赓,陈建庭. 低强度高频率振动对成骨细胞生物学特性的影响[J]. 中国临床解剖学杂志, 2018, 36(2): 165-173.

ZHA Ding-sheng, DENG Xuan-geng, CHEN Jian-ting. Effects of low magtitude and high frequency vibration on the biological characteristics of osteoblasts[J]. Chinese Journal Of Clinical Anatomy, 2018, 36(2): 165-173.

参考文献

[1] Phetfong J, Sanvoranart T, Nartprayut K, et al. Osteoporosis: the current status of mesenchymal stem cell-based therapy[J]. Cell Mol Biol Lett, 2016, 12; 21:12.
[2] 邵雄杰, 罗春晓, 陈郁鲜. 脉冲电磁场对绝经后骨质疏松症的疗效分析[J]. 南方医科大学学报, 2008, 28(9): 1577-1578.
[3] McMillan LB, Zengin A, Ebeling PR, et al. Prescribing physical activity for the prevention and treatment of osteoporosis in older adults[J]. Healthcare (Basel), 2017 , 5(4). pii: E85.
[4] Zha DS, Zhu QA, Pei WW, et al. Does whole-body vibration with alternative tilting increase bone mineral density and change bone metabolism in senior people? [J]. Aging Clin Exp Res, 2012, 24(1):28-36.
[5] 郑锦畅，陈建庭，裴卫卫，等.复合振动仪治疗骨质疏松对骨代谢影响6 个月临床观察[J]. 中国骨质疏松杂志, 2010, 16(3):197-202,180.
[6] 邓轩赓, 陈建庭, 查丁胜. 低强度复合振动治疗去卵巢老龄大鼠骨质疏松实验研究[J]. 中国运动医学杂志, 2011, 30(3):265-269, 274.
[7] 裴卫卫,陈建庭,郑锦畅,等.复合振动仪治疗原发骨质疏松症的短期临床疗效观察[J]. 中国骨质疏松杂志, 2009, 15(9):657-660.
[8] Wu SH, Zhong ZM, Chen JT.Low-magnitude high-frequency vibration inhibits RANKL-induced osteoclast differentiation of RAW264.7 cells[J]. Int J Med Sci, 2012, 9(9):801-7.
[9] Cerciello S, Rossi S, Visonà E, et al.Clinical applications of vibration therapy in orthopaedic practice[J]. Muscles Ligaments Tendons J, 2016 , 6(1):147-156.
[10]Papachroni KK, Karatzas DN, Papavassiliou KA, et al. Mechanotransduction in osteoblast regulation and bone disease[J]. Trends Mol Med, 2009, 15(5): 208-216.
[11]Prisby RD, Lafage-Proust MH, Malaval L, et al. Effects of whole body vibration on the skeleton and other organ systems in man and animal models: what we know and what we need to know[J]. Ageing Res Rev, 2008, 7(4): 319-329.
[12]查丁胜, 陈建庭, 邓轩庚, 等. 不同频率振动应变对成骨细胞增殖及分化能力的影响[J]. 中国骨质疏松杂志, 2008, 14(5): 303-307,312.
[13]Prè D, Ceccarelli G, Benedetti L, et al. Effects of low-amplitude, high-frequency vibrations on proliferation and differentiation of SAOS-2 human osteogenic cell line[J]. Tissue Eng Part C Methods, 2009, 15(4): 669-679.
[14]Patel MJ, Chang KH, Sykes MC, et al. Low magnitude and high frequency mechanical loading prevents decreased bone formation responses of 2T3 preosteoblasts[J]. J Cell Biochem, 2009, 106(2): 306-316.
[15]Grimaud E, Soubigou L, Couillaud S, et al. Receptor activator of nuclear factor kappaB ligand (RANKL)/osteoprotegerin (OPG) ratio is increased in severe osteolysis[J]. Am J Pathol, 2003, 163(5): 2021-2031.
[16]Saunders MM, Taylor AF, Du C, et al. Mechanical stimulation effects on functional end effectors in osteoblastic MG-63 cells[J]. J Biomech, 2006, 39(8): 1419-1427.
[17]Tang L, Lin Z, Li YM. Effects of different magnitudes of mechanical strain on Osteoblasts in vitro[J]. Biochem Biophys Res Commun, 2006, 344(1): 122-128.
[18]Lau E, Al-Dujaili S, Guenther A, et al. Effect of low-magnitude, high-frequency vibration on osteocytes in the regulation of osteoclasts[J]. Bone, 2010, 46(6): 1508-1515.
[19]Pagani F, Francucci CM, Moro L. Markers of bone turnover: biochemical and clinical perspectives[J]. J Endocrinol Invest, 2005, 28(10 Suppl): 8-13.
[20]钟招明. 生长分化因子-5诱导人黄韧带细胞成骨分化的作用及机制[D]. 广州：南方医科大学博士学位论文, 2009.
[21]Jadlowiec J, Koch H, Zhang X, et al. Phosphophoryn regulates the gene expression and differentiation of NIH3T3, MC3T3-E1, and human mesenchymal stem cells via the integrin/MAPK signaling pathway[J]. J Biol Chem, 2004, 279(51): 53323-53330.
[22]Xiao Y, Haase H, Young WG, et al. Development and transplantation of a mineralized matrix formed by osteoblasts in vitro for bone regeneration[J]. Cell Transplant, 2004, 13(1): 15-25.
[23]李志香, 张春林, 谈诚. 30Hz全身振动对骨质疏松的影响[J]. 航天医学与医学工程, 2007, 20(2):116-119.
[24]金凤羽, 阮祥燕. 机械振动治疗绝经后女性膝骨性关节炎[J]. 中国组织工程研究与临床康复, 2007, 11(40): 8099-8102.
[25]李玮, 钟菁, 徐惠敏, 等. 全身振动对绝经后肥胖妇女肌力及抗骨折能力的影响[J]. 重庆医科大学学报, 2008, 33(3): 367-369, 375.
[26]Dharmapatni AASSK, Algate K, Coleman R, et al.Osteoclast-associated receptor (OSCAR) distribution in the synovial tissues of patients with active ra and TNF-α and RANKL regulation of expression by osteoclasts in vitro[J]. Inflammation, 2017, 40(5):1566-1575.
[27]Kunnen SJ, Leonhard WN, Semeins C, et al. Fluid shear stress-induced TGF-β/ALK5 signaling in renal epithelial cells is modulated by MEK1/2[J]. Cell Mol Life Sci, 2017, 74(12): 2283-2298.
[28]Zhai Y, Li Y, Wang Y, et al. Psoralidin, a prenylated coumestan, as a novel anti-osteoporosis candidate to enhance bone formation of osteoblasts and decrease bone resorption of osteoclasts[J]. Eur J Pharmacol, 2017, 801:62-71.
[29]Scheiner S, Pivonka P, Hellmich C. Poromicromechanics reveals that physiological bone strains induce osteocyte-stimulating lacunar pressure[J]. Biomech Model Mechanobiol, 2016, 15(1): 9-28.