骨桥蛋白对巨噬细胞极化影响的体外实验研究
|
摘要
目的:探究外源性骨桥蛋白(osteopontin,OPN)在体外对巨噬细胞极化为M1型或M2型的影响。方法:体外培养小鼠RAW264.7细胞,进行以下分组实验:(1)OPN浓度梯度实验:设定对照组,实验组加入不同浓度OPN(0.1、0.5、1.0 mg/L);(2)M1型极化实验:设定对照组、脂多糖(lipopolysaccharide, LPS)组、OPN组、LPS+OPN组;(3)M2型极化实验:设定对照组、白细胞介素-4(interleukin-4, IL-4)组、OPN组、IL-4+OPN组。培养24 h后,实时荧光定量聚合酶链反应(quantitative real-time PCR, qRT-PCR)分析M1型基因诱导性一氧化氮合酶(inducible nitric oxide synthase, iNOS)、肿瘤坏死因子-α(tumor necrosis factorα, TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)及M2型基因CD206、精氨酸酶-1(arginase-1, Arg-1)、白细胞介素-10(interleukin-10, IL-10)的mRNA表达;Western blot检测iNOS、Arg-1的蛋白表达;免疫荧光检测iNOS、CD206的表达。结果:qRT-PCR显示,与对照组比较,OPN组的M1型基因iNOS、TNF-α、IL-1β及M2型基因CD206、Arg-1、IL-10的mRNA表达均上调(P<0.05),并呈浓度依赖关系;LPS+OPN组的M1型基因表达上调最高,IL-4+OPN组的M2型基因表达上调最高(P<0.05);Western blot、免疫荧光与qRT-PCR结果一致。结论:OPN作为一种多效性细胞因子,可能促进巨噬细胞向M1型或M2型极化,并与极化诱导剂LPS和IL-4具有潜在的协同促进作用。
Objective: To study the effect of exogenous osteopontin(OPN) on the polarization of macrophage in vitro. Methods: Mouse RAW264.7 macrophages were cultured in vitro, and divided into groups as follows:(1) OPN dose-dependent experiment: the experimental group were treated with 0.1,0.5,1.0 mg/L OPN respectively in vitro;(2) M1 polarization experiment: set control group, LPS group, OPN group, LPS+OPN group;(3) M2 polarization experiment: there were control group, IL-4 group, OPN group, and IL-4+OPN group. After stimulated for 24 hours, quantitative real-time PCR(qRT-PCR) was used to measure mRNA levels of M1-type genes inducible nitric oxide synthase(iNOS), tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and M2-type genes CD206, Arginase-1(Arg-1), and interleukin-10(IL-10). Western blot was used to assay iNOS and Arg-1 protein. The expression of iNOS and CD206 was detected by immunofluorescence assay(IFA). Results: mRNA levels of M1 and M2-type genes both increased significantly in a dose-dependent manner on OPN group(P<0.05). The expression of M1-type genes was the highest on LPS+OPN group, while expression of M2-type genes was the highest on IL-4+OPN group(P<0.05). The Western blot and IFA results were consistent with qRT-PCR results. Conclusion: OPN may induce M1 or M2 polarization of macrophage, and has synergetic effect with LPS and IL-4, as a pleiotropic cytokine.
Objective: To study the effect of exogenous osteopontin(OPN) on the polarization of macrophage in vitro. Methods: Mouse RAW264.7 macrophages were cultured in vitro, and divided into groups as follows:(1) OPN dose-dependent experiment: the experimental group were treated with 0.1,0.5,1.0 mg/L OPN respectively in vitro;(2) M1 polarization experiment: set control group, LPS group, OPN group, LPS+OPN group;(3) M2 polarization experiment: there were control group, IL-4 group, OPN group, and IL-4+OPN group. After stimulated for 24 hours, quantitative real-time PCR(qRT-PCR) was used to measure mRNA levels of M1-type genes inducible nitric oxide synthase(iNOS), tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and M2-type genes CD206, Arginase-1(Arg-1), and interleukin-10(IL-10). Western blot was used to assay iNOS and Arg-1 protein. The expression of iNOS and CD206 was detected by immunofluorescence assay(IFA). Results: mRNA levels of M1 and M2-type genes both increased significantly in a dose-dependent manner on OPN group(P<0.05). The expression of M1-type genes was the highest on LPS+OPN group, while expression of M2-type genes was the highest on IL-4+OPN group(P<0.05). The Western blot and IFA results were consistent with qRT-PCR results. Conclusion: OPN may induce M1 or M2 polarization of macrophage, and has synergetic effect with LPS and IL-4, as a pleiotropic cytokine.
引文
[1] Kzhyshkowska J,Gudima A,Riabov V,et al.Macrophage responses to implants:prospects for personalized medicine [J].J Leukoc Biol,2015,98(6):953-962.
[2] Martinez FO,Gordon S.The M1 and M2 paradigm of macrophage activation:time for reassessment [J].F1000Prime Rep,2014,6:13.
[3] Ivashkiv LB.Epigenetic regulation of macrophage polarization and function [J].Trends Immunol,2013,34(5):216-223.
[4] Murray PJ,Allen JE,Biswas SK,et al.Macrophage activation and polarization:nomenclature and experimental guidelines [J].Immunity,2014,41(1):14-20.
[5] Morris AH,Kyriakides TR.Matricellular proteins and biomaterials [J].Matrix Biol,2014,37:183-191.
[6] 符策广,刘洁,夏海斌.骨桥蛋白的生物学特性及在骨改建中的作用[J].临床口腔医学杂志,2012,28(8):506-508.
[7] Icer MA,Gezmen-Karadag M.The multiple functions and mechanisms of osteopontin [J].Clin Biochem,2018,59:17-24.
[8] Mckee MD,Pedraza CE,Kaartinen MT.Osteopontin and wound healing in bone [J].Cells Tissues Organs,2011,194(2-4):313-319.
[9] Makishi S,Saito K,Ohshima H.Osteopontin-deficiency disturbs direct osteogenesis in the process of achieving osseointegration following immediate placement of endosseous implants [J].Clin Implant Dent Relat Res,2017,19(3):496-504.
[10] Singh A,Gill G,Kaur H,et al.Role of osteopontin in bone remodeling and orthodontic tooth movement:a review [J].Prog Orthod,2018,19(1):18.
[11] 史欣,张鹏飞,袁梦桐,等.骨桥蛋白对人牙髓干细胞成骨分化的旁分泌作用[J].口腔医学研究,2014,30(11):1058-1061.
[12] Parisi L,Gini E,Baci D,et al.Macrophage polarization in chronic inflammatory diseases:killers or builders [J].J Immunol Res,2018,2018:8917804.
[13] Heilmann K,Hoffmann U,Witte E,et al.Osteopontin as two-sided mediator of intestinal inflammation [J].J Cell Mol Med,2009,13(6):1162-1174.
[14] Yang H,Ye X,Zhang X,et al.Intracellular osteopontin negatively regulates toll-like receptor 4-mediated inflammatory response via regulating GSK3β and 4EBP1 phosphorylation [J].Cytokine,2018,108:89-95.
[15] Many GM,Yokosaki Y,Uaesoontrachoon K,et al.OPN-a induces muscle inflammation by increasing recruitment and activation of pro-inflammatory macrophages [J].Exp Physiol,2016,101(10):1285-1300.
[16] Arango Duque G,Descoteaux A.Macrophage cytokines:involvement in immunity and infectious diseases [J].Front Immunol,2014,5:491.
[17] 李明政,肖宇,吴珍珍,等.巨噬细胞在材料诱导成骨中作用的研究进展[J].口腔医学研究,2017,33(10):1123-1126.
[18] Mosser DM,Edwards JP.Exploring the full spectrum of macrophage activation [J].Nat Rev Immunol,2008,8(12):958-969.
[19] Brown BN,Ratner BD,Goodman SB,et al.Macrophage polarization:An opportunity for improved outcomes in biomaterials and regenerative medicine [J].Biomaterials,2012,33(15):3792-3802.
[2] Martinez FO,Gordon S.The M1 and M2 paradigm of macrophage activation:time for reassessment [J].F1000Prime Rep,2014,6:13.
[3] Ivashkiv LB.Epigenetic regulation of macrophage polarization and function [J].Trends Immunol,2013,34(5):216-223.
[4] Murray PJ,Allen JE,Biswas SK,et al.Macrophage activation and polarization:nomenclature and experimental guidelines [J].Immunity,2014,41(1):14-20.
[5] Morris AH,Kyriakides TR.Matricellular proteins and biomaterials [J].Matrix Biol,2014,37:183-191.
[6] 符策广,刘洁,夏海斌.骨桥蛋白的生物学特性及在骨改建中的作用[J].临床口腔医学杂志,2012,28(8):506-508.
[7] Icer MA,Gezmen-Karadag M.The multiple functions and mechanisms of osteopontin [J].Clin Biochem,2018,59:17-24.
[8] Mckee MD,Pedraza CE,Kaartinen MT.Osteopontin and wound healing in bone [J].Cells Tissues Organs,2011,194(2-4):313-319.
[9] Makishi S,Saito K,Ohshima H.Osteopontin-deficiency disturbs direct osteogenesis in the process of achieving osseointegration following immediate placement of endosseous implants [J].Clin Implant Dent Relat Res,2017,19(3):496-504.
[10] Singh A,Gill G,Kaur H,et al.Role of osteopontin in bone remodeling and orthodontic tooth movement:a review [J].Prog Orthod,2018,19(1):18.
[11] 史欣,张鹏飞,袁梦桐,等.骨桥蛋白对人牙髓干细胞成骨分化的旁分泌作用[J].口腔医学研究,2014,30(11):1058-1061.
[12] Parisi L,Gini E,Baci D,et al.Macrophage polarization in chronic inflammatory diseases:killers or builders [J].J Immunol Res,2018,2018:8917804.
[13] Heilmann K,Hoffmann U,Witte E,et al.Osteopontin as two-sided mediator of intestinal inflammation [J].J Cell Mol Med,2009,13(6):1162-1174.
[14] Yang H,Ye X,Zhang X,et al.Intracellular osteopontin negatively regulates toll-like receptor 4-mediated inflammatory response via regulating GSK3β and 4EBP1 phosphorylation [J].Cytokine,2018,108:89-95.
[15] Many GM,Yokosaki Y,Uaesoontrachoon K,et al.OPN-a induces muscle inflammation by increasing recruitment and activation of pro-inflammatory macrophages [J].Exp Physiol,2016,101(10):1285-1300.
[16] Arango Duque G,Descoteaux A.Macrophage cytokines:involvement in immunity and infectious diseases [J].Front Immunol,2014,5:491.
[17] 李明政,肖宇,吴珍珍,等.巨噬细胞在材料诱导成骨中作用的研究进展[J].口腔医学研究,2017,33(10):1123-1126.
[18] Mosser DM,Edwards JP.Exploring the full spectrum of macrophage activation [J].Nat Rev Immunol,2008,8(12):958-969.
[19] Brown BN,Ratner BD,Goodman SB,et al.Macrophage polarization:An opportunity for improved outcomes in biomaterials and regenerative medicine [J].Biomaterials,2012,33(15):3792-3802.