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中华口腔医学研究杂志(电子版) ›› 2018, Vol. 12 ›› Issue (01) : 19 -25. doi: 10.3877/cma.j.issn.1674-1366.2018.01.004

所属专题: 文献

基础研究

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
陈倩莹1, 高雳1, 王盼盼1, 张驰1, 李希庭1, 赵川江1,()   
  1. 1. 510055 广州,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
  • 收稿日期:2017-09-28 出版日期:2018-02-01
  • 通信作者: 赵川江
  • 基金资助:
    广东省科技计划(2016A020215178); 广东省科技发展专项资金(2016A030310214)

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

Qianying Chen1, Li Gao1, Panpan Wang1, Chi Zhang1, Xiting Li1, Chuanjiang Zhao1,()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2017-09-28 Published:2018-02-01
  • Corresponding author: Chuanjiang Zhao
  • About author:
    Corresponding author:Zhao Chuanjiang,Email:
引用本文:

陈倩莹, 高雳, 王盼盼, 张驰, 李希庭, 赵川江. 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[J/OL]. 中华口腔医学研究杂志(电子版), 2018, 12(01): 19-25.

Qianying Chen, Li Gao, Panpan Wang, Chi Zhang, Xiting Li, Chuanjiang Zhao. The effect of interleukin-22 on the expression of RANKL and OPG in human periodontal ligament fibroblasts[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2018, 12(01): 19-25.

目的

探讨白细胞介素22(IL-22)对人牙周膜成纤维细胞(hPDLF)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hPDLF,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/mL)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/mL IL-22作用于hPDLF 72 h,实时荧光定量聚合酶链反应(PCR)检测RANKLOPG mRNA的表达;随后选择最佳刺激浓度10 ng/mL IL-22,与1 μg/mL Pg-LPS分别或协同刺激hPDLF 72 h,实时荧光定量PCR和蛋白质免疫印迹法(Western blot)检测RANKLOPG mRNA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/mL IL-22刺激hPDLF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/mL IL-22均可上调hPDLF RANKL mRNA表达(F= 32.88,P<0.05),但是对OPG mRNA表达无明显影响(F= 0.719,P= 0.555);10 ng/mL组和25 ng/mL组上调RANKL mRNA表达较5 ng/mL组显著(P<0.05);1 μg/mL Pg-LPS亦可显著上调hPDLF RANKL mRNA和蛋白水平的表达;而10 ng/mL IL-22与1 μg/mL Pg-LPS协同作用下RANKL mRNA和蛋白表达可进一步显著上调(FmRNA= 36.67,F蛋白= 41.24,P<0.05),但是对OPG的表达无明显影响(FmRNA= 0.652,P= 0.593;F蛋白= 1.271,P= 0.313)。

结论

IL-22可通过影响hPDLF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 1 μg/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 1 μg/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.

表1 实时荧光定量聚合酶链反应合成引物序列
图2 人牙周膜成纤维细胞免疫组化染色结果 图2A为人牙周膜成纤维细胞抗波形丝蛋白染色阳性,细胞浆呈棕黄色;图2B为人牙周膜成纤维细胞抗角蛋白染色阴性,细胞浆不着色
表2 不同浓度白细胞介素22(IL-22)对人牙周膜成纤维细胞(hPDLF)的毒性影响(%, ± s
图3 不同浓度白细胞介素22(IL-22)处理人牙周膜成纤维细胞(hPDLF)后各组相对细胞活力统计图
图4 白细胞介素22(IL-22)对人牙周膜成纤维细胞(hPDLF)核因子κB受体活化因子配体(RANKL)、骨保护素(OPG)mRNA表达的影响
图5 白细胞介素22(IL-22)、牙龈卟啉单胞菌脂多糖(Pg-LPS)协同对人牙周膜成纤维细胞(hPDLF)核因子κB受体活化因子配体(RANKL)、骨保护素(OPG)mRNA表达的影响
图6 白细胞介素22(IL-22)、牙龈卟啉单胞菌脂多糖(Pg-LPS)协同刺激对人牙周膜成纤维细胞(hPDLF)核因子κB受体活化因子配体(RANKL)、骨保护素(OPG)蛋白表达的影响
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