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中华口腔医学研究杂志(电子版) ›› 2017, Vol. 11 ›› Issue (06) : 333 -340. doi: 10.3877/cma.j.issn.1674-1366.2017.06.003

所属专题: 文献

基础研究

牙龈卟啉单胞菌脂多糖诱导人牙髓细胞炎症反应中微小RNA的表达谱变化
莫泽欢1, 徐琼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:2017-08-17 出版日期:2017-12-01
  • 通信作者: 徐琼
  • 基金资助:
    国家自然科学基金(81771058)

The expression profile of microRNA in Porphyromonas gingivalis lipopolysaccharide induced inflammation in human dental pulp cells

Zehuan Mo1, Qiong Xu1,()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2017-08-17 Published:2017-12-01
  • Corresponding author: Qiong Xu
  • About author:
    Corresponding author: Xu Qiong, Email:
引用本文:

莫泽欢, 徐琼. 牙龈卟啉单胞菌脂多糖诱导人牙髓细胞炎症反应中微小RNA的表达谱变化[J/OL]. 中华口腔医学研究杂志(电子版), 2017, 11(06): 333-340.

Zehuan Mo, Qiong Xu. The expression profile of microRNA in Porphyromonas gingivalis lipopolysaccharide induced inflammation in human dental pulp cells[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2017, 11(06): 333-340.

目的

研究牙龈卟啉单胞菌脂多糖(Pg-LPS)诱导人牙髓细胞(hDPC)炎症反应中微小RNA(miRNA)表达谱的变化。

方法

体外分离培养hDPC,10 μg/ml Pg-LPS处理hDPC 0、3、6、12、24 h,实时荧光定量聚合酶链反应(PCR)和ELISA检测炎症因子白细胞介素6(IL-6)、IL-8 mRNA及蛋白表达量。Illumina HiSeqTM 2500测序检测Pg-LPS刺激前后miRNA表达模式,筛选差异miRNA并预测其靶基因,对靶基因进行KEGG通路和GO功能富集分析。采用t检验或单因素方差分析等进行统计分析。

结果

Pg-LPS刺激hDPC,IL-6和IL-8的mRNA表达量及细胞上清蛋白含量显著升高,其中IL-6IL-8 mRNA在3 h表达升高最明显,分别为(7.4 ± 1.1)、(61.6 ± 20.8)倍,差异有统计学意义(tIL-6=-9.268,PIL-6 = 0.001;tIL-8=-5.047,PIL-8= 0.037)。IL-6和IL-8的分泌呈时间依赖性增加,24 h时含量分别为(44 ± 19)、(4016 ± 670)pg/ml,差异有统计学意义(tIL-6=-3.621,PIL-6= 0.022;tIL-8=-4.902,PIL-8= 0.008)。hDPC经LPS刺激后共检测出680个miRNA表达有变化,其中2个下调的miRNA和18个上调的miRNA为差异miRNA,功能富集分析显示差异miRNA参与调控多条炎症或损伤修复相关的信号通路如Wnt信号通路、TGF-β信号通路、PI3K-Akt信号通路、MAPK信号通路等。

结论

Pg-LPS可诱导hDPC发生炎症反应,此过程中miRNA表达谱发生变化,提示miRNA可能参与Pg-LPS诱导的hDPC炎症反应。

Objective

To investigate the microRNA (miRNA) expression profile in the inflammation of human dental pulp cells (hDPCs) induced by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) .

Methods

After isolated and cultured in vitro, the hDPCs were stimulated with Pg-LPS (10 μg/ml) for 0, 3, 6, 12, 24 h. The expression of inflammatory cytokines (IL-6, IL-8) were evaluated by reverse-transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) . Meanwhile Illumina HiSeqTM 2500 sequencing technology was used for investigating differentially expressed miRNAs. Bioinformatic analysis was applied for predicting the target genes of miRNAs and for Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis. Data were analyzed using t test or One-Way ANOVA.

Results

The mRNA expression of IL-6 and IL-8 were increased by (7.4 ± 1.1) , (61.6 ± 20.8) times respectively at 3 h with the peak by Pg-LPS (tIL-6=-9.268, PIL-6= 0.001; tIL-8=-5.047, PIL-8= 0.037) . The protein expression of IL-6 and IL-8 were upregulated by Pg-LPS in a time dependent manner with the concentration of (44 ± 19) , (4016 ± 670) pg/ml at 24 h (tIL-6=-3.621, PIL-6= 0.022; tIL-8=-4.902, PIL-8= 0.008) . A total of 680 miRNAs were detected with expression changed after stimulated with Pg-LPS (10 μg/ml) , and 2 down-regulated miRNAs and 18 up-regulated miRNAs were screening as differentially expressed miRNAs. Gene function enrichment analysis showed that differentially expressed miRNAs were involed in inflammatory related pathway such as Wnt signal pathway, TGF-β signal pathway, PI3K-Akt signal pathway, MAPK signal pathway.

Conclusion

Our data identified differential expression of miRNAs after hDPCs treated with Pg-LPS, indicating miRNAs may involed in the inflammation in hDPCs induced by Pg-LPS.

表1 实时荧光定量PCR引物序列
图1 CCK8法检测不同浓度脂多糖对人牙髓细胞增殖活性的影响
图2 实时荧光定量PCR检测脂多糖刺激人牙髓细胞中IL-6IL-8 mRNA表达情况
图3 双抗体夹心ELISA法检测脂多糖刺激人牙髓细胞中IL-6、IL-8的上清蛋白含量
图4 脂多糖刺激人牙髓细胞差异miRNA的表达情况
图5 差异miRNA的靶基因功能富集分析
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