牙周炎患者自身组织核酸对小鼠巨噬细胞中NLRP3 mRNA表达的影响及其意义
|
摘要
目的:检测牙周炎患者自身组织核酸刺激小鼠巨噬细胞后炎性相关因子NOD样受体蛋白3(NLRP3)、半胱氨酸天冬氨酸特异蛋白酶1 (caspase-1)、白细胞介素1β(IL-1β)和白细胞介素18 (IL-18)mRNA表达水平,探讨牙周炎患者自身组织核酸对小鼠巨噬细胞中炎性相关因子的作用。方法:于慢性牙周炎患者翻瓣术中采集炎性牙周组织、于牙周组织健康患者冠延长术中采集健康牙周组织,分别提取组织总RNA逆转录成cDNA (cDNA质量浓度为1mg·L~(-1))。培养小鼠巨噬细胞RAW264.7,分为对照组和实验组,分别加入上述提取的健康牙周组织cDNA (健康牙周组织)和炎症牙周组织cDNA (炎症牙周组织)共孵育。共孵育4、6和8h后,实时定量聚合酶链反应(RT-PCR)法检测各组RAW264.7细胞中NLRP3、caspase-1、IL-1β和IL-18mRNA表达水平。结果:显微镜下观察,小鼠巨噬细胞RAW264.7生长状态良好,细胞形态似圆形和多边形,胞浆透明,胞体丰满。与对照组比较,实验组在4、6和8h时RAW264.7细胞中NLRP3、caspase-1、IL-1β和IL-18mRNA表达水平明显升高(P<0.05或P<0.01),6h时实验组RAW264.7细胞中NLRP3、caspase-1、IL-1β和IL-18mRNA表达水平最高。结论:牙周炎患者自身组织核酸可促进小鼠巨噬细胞中炎性相关因子mRNA表达,牙周炎患者自身组织核酸对小鼠巨噬细胞的活化具有免疫调节作用。
Objective:To detect the mRNA expression levels of inflammatory-related factors NLRP3,caspase-1,IL-1β,and IL-18 in the murine macrophages infected by periodontitis patient's own tissue nucleic acid,and to discuss the effects of periodontitis patient's own tissue nucleic acid on the inflammation-related factors in the macrophages.Methods:The inflammatory periodontal tissue samples were collected during periodontal flap surgery of the chronic periodontitis patients,and the healthy periodontal tissue samples were collected from the patients without any periodontal diseases undergoing crown lengthening surgery.Then the total RNA from gingival tissue was extracted and reversely transcribed into cDNA.The cultured mouse macrophages RAW264.7 were divided into control group and experiment group,then the healthy periodontal tissue cDNA and inflammatory periodontal tissue cDNA(the cDNA at a concentration of 1 mg·L~(-1))were added into the RAW264.7 cells,respectively.Real-time PCR was used to detect the mRNA expression levels of NLRP3,Caspase-1,IL-1β,and IL-18 in the macrophages in various groups at 4,6 and 8 hafter incubation.Results:The microscope observation showed that the mouse macrophages RAW264.7 grew well with round and polygon shapes,clear cytoplasm,and full cell body.Compared with control group,the expression levels of NLRP3,Caspase-1,IL-1β,and IL-18 mRNA in the RAW264.7 cells in experiment group at 4,6,and 8 hwere increased significantly(P<0.05 or P<0.01),and the expression levels of NLRP3,Caspase-1,IL-1β,and IL-18 mRNA in RAW264.7 cells at 6 hin experiment group were the highest.Conclusion:The periodontitis patient's own tissue nucleic acids can promote the mRNA expressions of inflammation-related factors in the RAW264.7 cells,suggesting that the periodontitis patient's own tissue nucleic acid has an immunomodulatory effect on the activation of RAW264.7 cells.
Objective:To detect the mRNA expression levels of inflammatory-related factors NLRP3,caspase-1,IL-1β,and IL-18 in the murine macrophages infected by periodontitis patient's own tissue nucleic acid,and to discuss the effects of periodontitis patient's own tissue nucleic acid on the inflammation-related factors in the macrophages.Methods:The inflammatory periodontal tissue samples were collected during periodontal flap surgery of the chronic periodontitis patients,and the healthy periodontal tissue samples were collected from the patients without any periodontal diseases undergoing crown lengthening surgery.Then the total RNA from gingival tissue was extracted and reversely transcribed into cDNA.The cultured mouse macrophages RAW264.7 were divided into control group and experiment group,then the healthy periodontal tissue cDNA and inflammatory periodontal tissue cDNA(the cDNA at a concentration of 1 mg·L~(-1))were added into the RAW264.7 cells,respectively.Real-time PCR was used to detect the mRNA expression levels of NLRP3,Caspase-1,IL-1β,and IL-18 in the macrophages in various groups at 4,6 and 8 hafter incubation.Results:The microscope observation showed that the mouse macrophages RAW264.7 grew well with round and polygon shapes,clear cytoplasm,and full cell body.Compared with control group,the expression levels of NLRP3,Caspase-1,IL-1β,and IL-18 mRNA in the RAW264.7 cells in experiment group at 4,6,and 8 hwere increased significantly(P<0.05 or P<0.01),and the expression levels of NLRP3,Caspase-1,IL-1β,and IL-18 mRNA in RAW264.7 cells at 6 hin experiment group were the highest.Conclusion:The periodontitis patient's own tissue nucleic acids can promote the mRNA expressions of inflammation-related factors in the RAW264.7 cells,suggesting that the periodontitis patient's own tissue nucleic acid has an immunomodulatory effect on the activation of RAW264.7 cells.
引文
[1]CHENG R,LIU W,ZHANG R,et al.Porphyromonas gingivalis-derived lipopolysaccharide combines hypoxia to induce caspase-1activation in periodontitis[J].Front Cell Infect Microbiol,2017,7:474.
[2]DEIGENDESCH N,ZYCHLINSKY A,MEISSNER F.Copper regulates the canonical NLRP3inflammasome[J].J Immunol,2018,200(5):1607-1617.
[3]YAMAGUCHI Y,KURITA-OCHIAI T,KOBAYASHI R,et al.Regulation of the NLRP3 inflammasome in Porphyromonas gingivalis-accelerated periodontal disease[J].Inflamm Res,2017,66(1):59-65.
[4]XUE F,SHU R,XIE Y.The expression of NLRP3,NLRP1and AIM2in the gingival tissue of periodontitis patients:RT-PCR study and immunohistochemistry[J].Arch Oral Biol,2015,60(6):948-958.
[5]JO E K,KIM J K,SHIN D M,et al.Molecular mechanisms regulating NLRP3inflammasome activation[J].Cell Mol Immunol,2016,13(2):148-159.
[6]GONG T,YANG Y,JIN T,et al.Orchestration of NLRP3inflammasome activation by ion fluxes[J].Trends Immunol,2018,39(5):393-406.
[7]NEWTON K,DIXIT V M.Signaling in innate immunity and inflammation[J].Cold Spring Harb Perspect Biol,2012,4(3):a006049.
[8]丁子清,申玉芹,周岳,等.牙周炎患者自身组织核酸内NLRP3mRNA表达水平测定[J].口腔医学研究,2015,31(7):681-683.
[9]丁子清,申玉芹,周岳,等.牙周炎患者自身组织核酸刺激小鼠巨噬细胞后对破骨相关因子mRNA表达的影响[J].华西口腔医学杂志,2015,33(2):192-196.
[10]ARMITAGE G C.Development of a classification system for periodontal diseases and conditions[J].Ann Periodontol,1999,4(1):1-6.
[11]刘雯,郭文洁,徐强,等.NLRP3炎症小体调控机制研究进展[J].药学学报,2016,51(10):1505-1512.
[12]NURMI K,KAREINEN I,VIRKANEN J,et al.Hemin and cobalt protoporphyrin inhibit NLRP3 inflammasome activation by enhancing autophagy:a novel mechanism of inflammasome regulation[J].J Innate Immun,2017,9(1):65-82.
[13]YEON S H,YANG G,LEE H E,et al.Oxidized phosphatidylcholine induces the activation of NLRP3inflammasome in macrophages[J].J Leukoc Biol,2017,101(1):205-215.
[14]MONTENEGRO RAUDALES J L,YOSHIMURA A,SMZ,et al.Dental calculus stimulates interleukin-1βsecretion by activating NLRP3 inflammasome in human and mouse phagocytes[J].PLoS ONE,2016,11(9):e0162865.
[15]BUI F Q,JOHNSON L,ROBERTS J,et al.Fusobacterium nucleatum infection of gingival epithelial cells leads to NLRP3inflammasome-dependent secretion of IL-1βand the danger signals ASC and HMGB1[J].Cell Microbiol,2016,18(7):970-981.
[16]CHENG R,FENG Y,ZHANG R,et al.The extent of pyroptosis varies in different stages of apical periodontitis[J].Biochim Biophys Acta,2018,1864(1):226-237.
[17]PERRI R,NARES S,ZHANG S,et al.MicroRNAmodulation in obesity and periodontitis[J].J Dent Res,2012,91(1):33-38.
[18]YU N,BARROS S P,ZHANG S,et al.Insulin response genes in different stages of periodontal disease[J].J Dent Res,2015,94(9Suppl):194S-200S.
[19]BEYER C,STEARNS N A,GIESSL A,et al.The extracellular release of DNA and HMGB1from Jurkat T cells during in vitro necrotic cell death[J].Innate Immun,2012,18(5):727-737.
[20]陈倩柔,卢顿朗,苗梦雨,等.下前牙松牙固定的生物力学分析[J].郑州大学学报:医学版,2018,53(2):245-250.
[21]吴冷,王骏,赵蕾,等.核苷酸结合寡聚化结构域样受体热蛋白结构域亚家族成员3炎症小体的活化调节与牙周疾病的关系[J].国际口腔医学杂志,2015,42(6):710-714.
[2]DEIGENDESCH N,ZYCHLINSKY A,MEISSNER F.Copper regulates the canonical NLRP3inflammasome[J].J Immunol,2018,200(5):1607-1617.
[3]YAMAGUCHI Y,KURITA-OCHIAI T,KOBAYASHI R,et al.Regulation of the NLRP3 inflammasome in Porphyromonas gingivalis-accelerated periodontal disease[J].Inflamm Res,2017,66(1):59-65.
[4]XUE F,SHU R,XIE Y.The expression of NLRP3,NLRP1and AIM2in the gingival tissue of periodontitis patients:RT-PCR study and immunohistochemistry[J].Arch Oral Biol,2015,60(6):948-958.
[5]JO E K,KIM J K,SHIN D M,et al.Molecular mechanisms regulating NLRP3inflammasome activation[J].Cell Mol Immunol,2016,13(2):148-159.
[6]GONG T,YANG Y,JIN T,et al.Orchestration of NLRP3inflammasome activation by ion fluxes[J].Trends Immunol,2018,39(5):393-406.
[7]NEWTON K,DIXIT V M.Signaling in innate immunity and inflammation[J].Cold Spring Harb Perspect Biol,2012,4(3):a006049.
[8]丁子清,申玉芹,周岳,等.牙周炎患者自身组织核酸内NLRP3mRNA表达水平测定[J].口腔医学研究,2015,31(7):681-683.
[9]丁子清,申玉芹,周岳,等.牙周炎患者自身组织核酸刺激小鼠巨噬细胞后对破骨相关因子mRNA表达的影响[J].华西口腔医学杂志,2015,33(2):192-196.
[10]ARMITAGE G C.Development of a classification system for periodontal diseases and conditions[J].Ann Periodontol,1999,4(1):1-6.
[11]刘雯,郭文洁,徐强,等.NLRP3炎症小体调控机制研究进展[J].药学学报,2016,51(10):1505-1512.
[12]NURMI K,KAREINEN I,VIRKANEN J,et al.Hemin and cobalt protoporphyrin inhibit NLRP3 inflammasome activation by enhancing autophagy:a novel mechanism of inflammasome regulation[J].J Innate Immun,2017,9(1):65-82.
[13]YEON S H,YANG G,LEE H E,et al.Oxidized phosphatidylcholine induces the activation of NLRP3inflammasome in macrophages[J].J Leukoc Biol,2017,101(1):205-215.
[14]MONTENEGRO RAUDALES J L,YOSHIMURA A,SMZ,et al.Dental calculus stimulates interleukin-1βsecretion by activating NLRP3 inflammasome in human and mouse phagocytes[J].PLoS ONE,2016,11(9):e0162865.
[15]BUI F Q,JOHNSON L,ROBERTS J,et al.Fusobacterium nucleatum infection of gingival epithelial cells leads to NLRP3inflammasome-dependent secretion of IL-1βand the danger signals ASC and HMGB1[J].Cell Microbiol,2016,18(7):970-981.
[16]CHENG R,FENG Y,ZHANG R,et al.The extent of pyroptosis varies in different stages of apical periodontitis[J].Biochim Biophys Acta,2018,1864(1):226-237.
[17]PERRI R,NARES S,ZHANG S,et al.MicroRNAmodulation in obesity and periodontitis[J].J Dent Res,2012,91(1):33-38.
[18]YU N,BARROS S P,ZHANG S,et al.Insulin response genes in different stages of periodontal disease[J].J Dent Res,2015,94(9Suppl):194S-200S.
[19]BEYER C,STEARNS N A,GIESSL A,et al.The extracellular release of DNA and HMGB1from Jurkat T cells during in vitro necrotic cell death[J].Innate Immun,2012,18(5):727-737.
[20]陈倩柔,卢顿朗,苗梦雨,等.下前牙松牙固定的生物力学分析[J].郑州大学学报:医学版,2018,53(2):245-250.
[21]吴冷,王骏,赵蕾,等.核苷酸结合寡聚化结构域样受体热蛋白结构域亚家族成员3炎症小体的活化调节与牙周疾病的关系[J].国际口腔医学杂志,2015,42(6):710-714.