白细胞介素17和牙周膜成纤维细胞在体外诱导破骨样细胞生成中的作用
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摘要
背景:研究表明,牙根吸收是正畸治疗过程中常见的并发症,而白细胞介素17可能通过牙周膜参与介导破骨细胞的分化和成熟从而引发牙根吸收。目的:探讨白细胞介素17和人牙周膜成纤维细胞在正畸炎性相关牙根吸收中的作用及其作用机制。方法:取4-6代人牙周膜成纤维细胞分别加入含白细胞介素17质量浓度为0或20μg/L的DMEM培养基预处理42 h,取细胞上清液加入50μg/L核因子κB受体活化因子配体(RANKL)分别作用于外周血单核细胞3,5,7,10 d,建立人牙周膜成纤维细胞和外周血单核细胞间接共培养系统。实验分为6组:(1)对照组;(2)外周血单核细胞+RANKL组;(3)外周血单核细胞+人牙周膜成纤维细胞组;(4)外周血单核细胞+人牙周膜成纤维细胞+白细胞介素17组;(5)外周血单核细胞+人牙周膜成纤维细胞+RANKL组;(6)外周血单核细胞+人牙周膜成纤维细胞+RANKL+白细胞介素17组。采用RT-qPCR法检测外周血单核细胞诱导生成破骨样细胞组织蛋白酶K、基质金属蛋白酶9、碳酸酐酶Ⅱm RNA的表达量,鬼笔环肽染色观察和计数破骨样细胞的细胞骨架中纤维肌动蛋白环的形态和数目。结果与结论:(1)RT-qPCR分析证实,与对照组相比,白细胞介素17、人牙周膜成纤维细胞和RANKL诱导的基质金属蛋白酶9、组织蛋白酶K和碳酸酐酶Ⅱ表达水平显著提高;(2)鬼笔环肽染色结果显示,与对照组相比,加白细胞介素17组或(和)加RANKL组肌动蛋白环阳性细胞数目显著增加(P <0.01);(3)结果提示,白细胞介素17可有效诱导破骨细胞前体分化为具有骨吸收能力的破骨样细胞,其具体机制是通过调节人牙周膜成纤维细胞,上调RANKL下调骨保护素来介导的。
BACKGROUND: Root resorption has been shown to be a common complication during orthodontic treatment, and interleukin-17 may mediate the differentiation and maturation of osteoclasts through periodontal ligament to induce root resorption. OBJECTIVE: To investigate the roles of interleukin-17 and human periodontal ligament fibroblasts in orthodontically induced inflammatory root resorption and the underlying mechanism. METHODS: Passage 4-6 human periodontal ligament fibroblasts were added to the DMEM medium containing interleukin-17 at concentration of 0 or 20 μg/L for 42 hours. Cell supernatant mixed with 50 μg/L RANKL was cultured with peripheral blood mononuclear cells for 3, 5, 7 and 10 days, respectively to establish a human periodontal ligament fibroblasts and peripheral blood mononuclear cells indirect co-culture system. There were six groups: control group, peripheral blood mononuclear cells+RANKL group, peripheral blood mononuclear cells+human periodontal ligament fibroblasts group, peripheral blood mononuclear cells+human periodontal ligament fibroblasts+interleukin-17 group, peripheral blood mononuclear cells + human periodontal ligament fibroblasts+RANKL group, and peripheral blood mononuclear cells+human periodontal ligament fibroblasts+RANKL+interleukin-17 group. The expression levels of cathepsin K, matrix metalloproteinase 9 and carbonic anhydrase II mRNA induced by peripheral blood mononuclear cells in osteoclast-like cells were detected by RT-qPCR. Phalloidin staining was used to observe and count the morphology and number of fibromuscular actin rings in the cytoskeleton of osteoclast-like cells. RESULTS AND CONCLUSIONS: RT-qPCR analysis confirmed that interleukin-17, human periodontal ligament fibroblasts and RANKL increased the expression levels of cathepsin K, matrix metalloproteinase 9 and carbonic anhydrase II compared with the control group. The results of phalloidin staining showed a significant increase in the number of actin-loop positive cells in the interleukin-17(+) or RANKL(+) groups compared with the control group(P < 0.01). To conclude, interleukin-17 can effectively induce osteoclast precursor differentiation into osteoclast-like cells with bone resorption ability, which may be mediated through upregulation of RANKL and downregulation of osteoprotegerin by human periodontal ligament fibroblasts.
BACKGROUND: Root resorption has been shown to be a common complication during orthodontic treatment, and interleukin-17 may mediate the differentiation and maturation of osteoclasts through periodontal ligament to induce root resorption. OBJECTIVE: To investigate the roles of interleukin-17 and human periodontal ligament fibroblasts in orthodontically induced inflammatory root resorption and the underlying mechanism. METHODS: Passage 4-6 human periodontal ligament fibroblasts were added to the DMEM medium containing interleukin-17 at concentration of 0 or 20 μg/L for 42 hours. Cell supernatant mixed with 50 μg/L RANKL was cultured with peripheral blood mononuclear cells for 3, 5, 7 and 10 days, respectively to establish a human periodontal ligament fibroblasts and peripheral blood mononuclear cells indirect co-culture system. There were six groups: control group, peripheral blood mononuclear cells+RANKL group, peripheral blood mononuclear cells+human periodontal ligament fibroblasts group, peripheral blood mononuclear cells+human periodontal ligament fibroblasts+interleukin-17 group, peripheral blood mononuclear cells + human periodontal ligament fibroblasts+RANKL group, and peripheral blood mononuclear cells+human periodontal ligament fibroblasts+RANKL+interleukin-17 group. The expression levels of cathepsin K, matrix metalloproteinase 9 and carbonic anhydrase II mRNA induced by peripheral blood mononuclear cells in osteoclast-like cells were detected by RT-qPCR. Phalloidin staining was used to observe and count the morphology and number of fibromuscular actin rings in the cytoskeleton of osteoclast-like cells. RESULTS AND CONCLUSIONS: RT-qPCR analysis confirmed that interleukin-17, human periodontal ligament fibroblasts and RANKL increased the expression levels of cathepsin K, matrix metalloproteinase 9 and carbonic anhydrase II compared with the control group. The results of phalloidin staining showed a significant increase in the number of actin-loop positive cells in the interleukin-17(+) or RANKL(+) groups compared with the control group(P < 0.01). To conclude, interleukin-17 can effectively induce osteoclast precursor differentiation into osteoclast-like cells with bone resorption ability, which may be mediated through upregulation of RANKL and downregulation of osteoprotegerin by human periodontal ligament fibroblasts.
引文
[1]Makrygiannakis MA,Kaklamanos EG,Athanasiou AE.Effects of systemic medication on root resorption associated with orthodontic tooth movement:a systematic review of animal studies.Eur J Orthod.2018 Jul 9.[Epub ahead of print].
[2]Yashin D,Dalci O,Almuzian M,et al.Markers in blood and saliva for prediction of orthodontically induced inflammatory root resorption:a retrospective case controlled-study.Prog Orthod.2017;18(1):27.
[3]孟冉冉,宋萌,潘劲松,等.正畸牙移动相关信号通路研究进展[J].现代生物医学进展,2015,15(20):3961-3913.
[4]Nakano Y,Yamaguchi M,Fujita S,et al.Expressions of RANKL/RANK and M-CSF/c-fms in root resorption lacunae in rat molar by heavy orthodontic force.Eur J Orthod.2011;33(4):335-343.
[5]李光辉,潘晓岗.参与正畸牙移动的相关因子研究进展[J].口腔医学,2015,35(6):508-512.
[6]Baloul SS.Osteoclastogenesis and Osteogenesis during Tooth Movement.Front Oral Biol.2016;18:75-79.
[7]Martins CA,Leyhausen G,Volk J,et al.Effects of alendronate on osteoclast formation and activity in vitro.J Endod.2015;41(1):45-49.
[8]Zhang J,Xu H,Han Z,et al.Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca(2+)-calcineurin-NFATc1signaling pathway.Biochem Biophys Res Commun.2017;482(2):289-295.
[9]van Heerden B,Kasonga A,Kruger MC,et al.Palmitoleic Acid Inhibits RANKL-Induced Osteoclastogenesis and Bone Resorption by Suppressing NF-kappaB and MAPK Signalling Pathways.Nutrients.Nutrients.2017;9(5).pii:E441.
[10]Feller L,Khammissa RA,Thomadakis G,et al.Apical External Root Resorption and Repair in Orthodontic Tooth Movement:Biological Events.Biomed Res Int.2016;2016:4864195.
[11]Hellsing E,Hammarstrom L.The hyaline zone and associated root surface changes in experimental orthodontics in rats:a light and scanning electron microscope study.Eur J Orthod.1996;18(1):11-18.
[12]Roscoe MG,Meira JB,Cattaneo PM.Association of orthodontic force system and root resorption:A systematic review.Am J Orthod Dentofacial Orthop.2015;147(5):610-626.
[13]Walsh MC,Kim N,Kadono Y,et al.Osteoimmunology:interplay between the immune system and bone metabolism.Annu Rev Immunol.2006;24:33-63.Review.
[14]ChesnéJ,Braza F,Mahay G,et al.IL-17 in severe asthma.Where do we stand?.Am J Respir Crit Care Med.2014;190(10):1094-1101.
[15]Adamopoulos IE,Suzuki E,Chao CC,et al.IL-17A gene transfer induces bone loss and epidermal hyperplasia associated with psoriatic arthritis.Ann Rheum Dis.2015;74(6):1284-1292.
[16]Ling Y,Puel A.IL-17 and infections.Actas Dermosifiliogr.2014;105 Suppl 1:34-40.
[17]Wang ZX,Yang L,Tan JY,et al.[Effects of T helper 1 cells and T helper 17 cells secreting cytokines on rat models of experimental periodontitis]Zhonghua Kou Qiang Yi Xue Za Zhi.2017;52(12):740-747..
[18]Gaffen SL.Structure and signalling in the IL-17 receptor family.Nat Rev Immunol.2009;9(8):556-567.
[19]Takahashi K,Azuma T,Motohira H,et al.The potential role of interleukin-17 in the immunopathology of periodontal disease.J Clin Periodontol.2005;32(4):369-374.
[20]Chen W,Wang J,Xu Z,et al.Apremilast Ameliorates Experimental Arthritis via Suppression of Th1 and Th17 Cells and Enhancement of CD4(+)Foxp3(+)Regulatory T Cells Differentiation.Front Immunol.2018;9:1662.
[21]Kotake S,Yago T,Kawamoto M,et al.Role of osteoclasts and interleukin-17 in the pathogenesis of rheumatoid arthritis:crucial'human osteoclastology'.J Bone Miner Metab.2012;30(2):125-135.
[22]Chitrapriya MN,Rao SR,Lavu V.Interleukin-17 and interleukin-18 levels in different stages of inflammatory periodontal disease.J Indian Soc Periodontol.2015;19(1):14-17.
[23]Nakano Y,Yamaguchi M,Shimizu M,et al.Interleukin-17 is involved in orthodontically induced inflammatory root resorption in dental pulp cells.Am J Orthod Dentofacial Orthop.2015;148(2):302-309.
[24]Sims NA,Martin TJ.Coupling Signals between the Osteoclast and Osteoblast:How are Messages Transmitted between These Temporary Visitors to the Bone Surface?.Front Endocrinol(Lausanne).2015;6:41.
[25]Wang Q,Yao L,Xu K,et al.Madecassoside inhibits estrogen deficiency-induced osteoporosis by suppressing RANKL-induced osteoclastogenesis.J Cell Mol Med.2018Oct 19.
[26]Kikuta J,Yamaguchi M,Shimizu M,et al.Notch signaling induces root resorption via RANKL and IL-6 from hPDL cells.J Dent Res.2015;94(1):140-147.
[27]de Vries TJ,Yousovich J,Schoenmaker T,et al.Tumor necrosis factor-alpha antagonist infliximab inhibits osteoclast formation of peripheral blood mononuclear cells but does not affect periodontal ligament fibroblast-mediated osteoclast formation.J Periodontal Res.2016;51(2):186-195.
[28]Matsuzaki K,Udagawa N,Takahashi N,et al.Osteoclast differentiation factor(ODF)induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures.Biochem Biophys Res Commun.1998;246(1):199-204.
[29]Cao H,Kou X,Yang R,et al.Force-induced Adrb2 in periodontal ligament cells promotes tooth movement.J Dent Res.2014;93(11):1163-1169.
[30]李琳娟,黎敏,彭娟敏,等.白细胞介素17对人牙周膜成纤维细胞表达RANKL、OPG的影响[J].重庆医学,2017,46(23):3177-3179.
[31]Ye S,Fujiwara T,Zhou J,et al.LIS1 Regulates Osteoclastogenesis through Modulation of M-SCF and RANKL Signaling Pathways and CDC42.Int J Biol Sci.2016;12(12):1488-1499.
[32]Bloemen V,Schoenmaker T,de Vries TJ,et al.Direct cell-cell contact between periodontal ligament fibroblasts and osteoclast precursors synergistically increases the expression of genes related to osteoclastogenesis.J Cell Physiol.2010;222(3):565-573.
[33]Weltman B,Vig KW,Fields HW,et al.Root resorption associated with orthodontic tooth movement:a systematic review.Am J Orthod Dentofacial Orthop.2010;137(4):462-476;discussion 12A.
[2]Yashin D,Dalci O,Almuzian M,et al.Markers in blood and saliva for prediction of orthodontically induced inflammatory root resorption:a retrospective case controlled-study.Prog Orthod.2017;18(1):27.
[3]孟冉冉,宋萌,潘劲松,等.正畸牙移动相关信号通路研究进展[J].现代生物医学进展,2015,15(20):3961-3913.
[4]Nakano Y,Yamaguchi M,Fujita S,et al.Expressions of RANKL/RANK and M-CSF/c-fms in root resorption lacunae in rat molar by heavy orthodontic force.Eur J Orthod.2011;33(4):335-343.
[5]李光辉,潘晓岗.参与正畸牙移动的相关因子研究进展[J].口腔医学,2015,35(6):508-512.
[6]Baloul SS.Osteoclastogenesis and Osteogenesis during Tooth Movement.Front Oral Biol.2016;18:75-79.
[7]Martins CA,Leyhausen G,Volk J,et al.Effects of alendronate on osteoclast formation and activity in vitro.J Endod.2015;41(1):45-49.
[8]Zhang J,Xu H,Han Z,et al.Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca(2+)-calcineurin-NFATc1signaling pathway.Biochem Biophys Res Commun.2017;482(2):289-295.
[9]van Heerden B,Kasonga A,Kruger MC,et al.Palmitoleic Acid Inhibits RANKL-Induced Osteoclastogenesis and Bone Resorption by Suppressing NF-kappaB and MAPK Signalling Pathways.Nutrients.Nutrients.2017;9(5).pii:E441.
[10]Feller L,Khammissa RA,Thomadakis G,et al.Apical External Root Resorption and Repair in Orthodontic Tooth Movement:Biological Events.Biomed Res Int.2016;2016:4864195.
[11]Hellsing E,Hammarstrom L.The hyaline zone and associated root surface changes in experimental orthodontics in rats:a light and scanning electron microscope study.Eur J Orthod.1996;18(1):11-18.
[12]Roscoe MG,Meira JB,Cattaneo PM.Association of orthodontic force system and root resorption:A systematic review.Am J Orthod Dentofacial Orthop.2015;147(5):610-626.
[13]Walsh MC,Kim N,Kadono Y,et al.Osteoimmunology:interplay between the immune system and bone metabolism.Annu Rev Immunol.2006;24:33-63.Review.
[14]ChesnéJ,Braza F,Mahay G,et al.IL-17 in severe asthma.Where do we stand?.Am J Respir Crit Care Med.2014;190(10):1094-1101.
[15]Adamopoulos IE,Suzuki E,Chao CC,et al.IL-17A gene transfer induces bone loss and epidermal hyperplasia associated with psoriatic arthritis.Ann Rheum Dis.2015;74(6):1284-1292.
[16]Ling Y,Puel A.IL-17 and infections.Actas Dermosifiliogr.2014;105 Suppl 1:34-40.
[17]Wang ZX,Yang L,Tan JY,et al.[Effects of T helper 1 cells and T helper 17 cells secreting cytokines on rat models of experimental periodontitis]Zhonghua Kou Qiang Yi Xue Za Zhi.2017;52(12):740-747..
[18]Gaffen SL.Structure and signalling in the IL-17 receptor family.Nat Rev Immunol.2009;9(8):556-567.
[19]Takahashi K,Azuma T,Motohira H,et al.The potential role of interleukin-17 in the immunopathology of periodontal disease.J Clin Periodontol.2005;32(4):369-374.
[20]Chen W,Wang J,Xu Z,et al.Apremilast Ameliorates Experimental Arthritis via Suppression of Th1 and Th17 Cells and Enhancement of CD4(+)Foxp3(+)Regulatory T Cells Differentiation.Front Immunol.2018;9:1662.
[21]Kotake S,Yago T,Kawamoto M,et al.Role of osteoclasts and interleukin-17 in the pathogenesis of rheumatoid arthritis:crucial'human osteoclastology'.J Bone Miner Metab.2012;30(2):125-135.
[22]Chitrapriya MN,Rao SR,Lavu V.Interleukin-17 and interleukin-18 levels in different stages of inflammatory periodontal disease.J Indian Soc Periodontol.2015;19(1):14-17.
[23]Nakano Y,Yamaguchi M,Shimizu M,et al.Interleukin-17 is involved in orthodontically induced inflammatory root resorption in dental pulp cells.Am J Orthod Dentofacial Orthop.2015;148(2):302-309.
[24]Sims NA,Martin TJ.Coupling Signals between the Osteoclast and Osteoblast:How are Messages Transmitted between These Temporary Visitors to the Bone Surface?.Front Endocrinol(Lausanne).2015;6:41.
[25]Wang Q,Yao L,Xu K,et al.Madecassoside inhibits estrogen deficiency-induced osteoporosis by suppressing RANKL-induced osteoclastogenesis.J Cell Mol Med.2018Oct 19.
[26]Kikuta J,Yamaguchi M,Shimizu M,et al.Notch signaling induces root resorption via RANKL and IL-6 from hPDL cells.J Dent Res.2015;94(1):140-147.
[27]de Vries TJ,Yousovich J,Schoenmaker T,et al.Tumor necrosis factor-alpha antagonist infliximab inhibits osteoclast formation of peripheral blood mononuclear cells but does not affect periodontal ligament fibroblast-mediated osteoclast formation.J Periodontal Res.2016;51(2):186-195.
[28]Matsuzaki K,Udagawa N,Takahashi N,et al.Osteoclast differentiation factor(ODF)induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures.Biochem Biophys Res Commun.1998;246(1):199-204.
[29]Cao H,Kou X,Yang R,et al.Force-induced Adrb2 in periodontal ligament cells promotes tooth movement.J Dent Res.2014;93(11):1163-1169.
[30]李琳娟,黎敏,彭娟敏,等.白细胞介素17对人牙周膜成纤维细胞表达RANKL、OPG的影响[J].重庆医学,2017,46(23):3177-3179.
[31]Ye S,Fujiwara T,Zhou J,et al.LIS1 Regulates Osteoclastogenesis through Modulation of M-SCF and RANKL Signaling Pathways and CDC42.Int J Biol Sci.2016;12(12):1488-1499.
[32]Bloemen V,Schoenmaker T,de Vries TJ,et al.Direct cell-cell contact between periodontal ligament fibroblasts and osteoclast precursors synergistically increases the expression of genes related to osteoclastogenesis.J Cell Physiol.2010;222(3):565-573.
[33]Weltman B,Vig KW,Fields HW,et al.Root resorption associated with orthodontic tooth movement:a systematic review.Am J Orthod Dentofacial Orthop.2010;137(4):462-476;discussion 12A.