白细胞介素22对人牙周膜成纤维细胞核因子κB受体活化因子配体、骨保护素表达的影响  

The effect of interleukin-22 on the expression of RANKL and OPG in human periodontal ligament fibroblasts

作　　者：陈倩莹 高雳[1] 王盼盼[1] 张驰[1] 李希庭[1] 赵川江[1] Chen Qianying;Gao Li;Wang Panpan;Zhang Chi;Li Xiting;Zhao Chuanjiang(Guanghua School of Stomatology,Hospital of Stomatology,Sun Yat.sen University,Guangdong Provincial Key Laboratory of Stomatology,Guangzhou 510055,China)

机构地区：[1]中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室,广州510055

出　　处：《中华口腔医学研究杂志（电子版）》2018年第1期19-25,共7页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：广东省科技计划(2016A020215178);广东省科技发展专项资金(2016A030310214)

摘　　要：目的探讨白细胞介素22(IL-22)对人牙周膜成纤维细胞(h PDLF)表达核因子κB受体活化因子配体(RANKL)、骨保护素(OPG)的影响;并进一步研究IL-22是否与牙龈卟啉单胞菌脂多糖(Pg-LPS)具有协同刺激作用。方法酶消化法结合组织块法原代培养h PDLF,免疫细胞化学染色法鉴定其来源;取第3~5代细胞给予不同浓度(0、5、10、25、50、100 ng/m L)IL-22刺激,培养24、48、72 h采用细胞计数试剂盒(CCK-8)进行细胞毒性实验;采用5、10、25 ng/m L IL-22作用于h PDLF 72 h,实时荧光定量聚合酶链反应(PCR)检测RANKL、OPG m RNA的表达;随后选择最佳刺激浓度10 ng/m LIL-22,与1μg/m L Pg-LPS分别或协同刺激h PDLF 72 h,实时荧光定量PCR和蛋白质免疫印迹法(Western blot)检测RANKL、OPG m RNA和蛋白水平的表达。采用单因素方差分析对数据进行统计分析。结果 50、100 ng/m L IL-22刺激h PDLF后,细胞活力明显下降(F24 h=15.17,F48 h=76.37,F72 h=24.409,P<0.05);5、10、25 ng/m L IL-22均可上调h PDLF RANKL m RNA表达(F=32.88,P<0.05),但是对OPG m RNA表达无明显影响(F=0.719,P=0.555);10 ng/m L组和25 ng/m L组上调RANKL m RNA表达较5 ng/m L组显著(P<0.05);1μg/m L Pg-LPS亦可显著上调h PDLF RANKL m RNA和蛋白水平的表达;而10 ng/m L IL-22与1μg/m L Pg-LPS协同作用下RANKL m RNA和蛋白表达可进一步显著上调(Fm RNA=36.67,F蛋白=41.24,P<0.05),但是对OPG的表达无明显影响(Fm RNA=0.652,P=0.593;F蛋白=1.271,P=0.313)。结论 IL-22可通过影响h PDLF表达RANKL/OPG参与牙周炎骨破坏,并且与Pg-LPS具有协同作用。Objective To investigate the effect of interleukin-22(IL-22)on the expression of RANKL and OPG in human periodontal ligament fibroblasts(hPDLFs),and further explore the synergistic effect of IL-22 and Porphyromonas gingivalis lipopolysaccharide(Pg-LPS).Methods Primary hPDLFs were cultured using combined methods of tissue explant and enzymatic digestion,then identified by immunocytochemical staining.Passage third-fifth cells were treated with different concentrations of IL-22(0~100 ng/mL),and cell counting kit-8(CCK-8)assay was used to detect the cytotoxicity at 24,48,72 h.5,10,25 ng/mL IL-22 were applied to hPDLFs,and the expression of RANKL and OPG mRNA were assessed by quantitative real-time polymerase chain reaction(qRT-PCR)at 72 h.Furthermore,cells were treated with 1μg/mL Pg-LPS alone,or together with 10 ng/mL IL-22 for 72 h.The expression of RANKL and OPG mRNA and their coding proteins were measured using qRT-PCR and Western blot.Data were evaluated by One-Way ANOVA.Results The 50 ng/mL and 100 ng/mL IL-22 significantly decreased cell viability of hPDLFs,comparing to the control group(F24 h=15.17,F48 h=76.37,F72 h=24.409,P<0.05).A number of concentrations of IL-22(5,10 and 25 ng/mL)up-regulated the mRNA expression of RANKL in hPDLFs(F=32.88,P<0.05),with 10,25 ng/mL more significantly than 5 ng/mL(P<0.05).However,the mRNA expression of OPG was not affected(F=0.719,P=0.555).Co-stimulation with 10 ng/mL IL-22 and 1μg/mL Pg-LPS enhanced both RANKL mRNA and protein expression comparing with those of IL-22 or Pg-LPS stimulation alone(FmRNA=36.67,Fprotein=41.24,P<0.05).OPG mRNA and protein were not changed compared with the control(FmRNA=0.652,P=0.593;Fprotein=1.271,P=0.313).Conclusions IL-22 up-regulates RANKL expression in hPDLFs,and has synergistic effect with Pg-LPS,suggesting that IL-22 may participate in periodontal bone destruction.

关 键 词：白细胞介素22 牙龈卟啉单胞菌 脂多糖类 核因子ΚB受体活化因子 配体 骨保护素 

分 类 号：R781.42[医药卫生—口腔医学]