Department of Morphology, Laboratory of Molecular Biology, Division of Histology, Piracicaba Dental School, University of Campinas-UNICAMP, Piracicaba, São Paulo, SP, Brazil.
J Clin Periodontol. 2011 Nov;38(11):975-83. doi: 10.1111/j.1600-051X.2011.01765.x. Epub 2011 Sep 7.
The objective of this study was to analyse the status of DNA methylation in the promoter region of the toll-like receptor (TLR)2 and TLR4 genes in gingival tissue samples from healthy subjects, smokers and non-smokers affected by chronic periodontitis.
Genomic DNA and total RNA were purified from gingival tissue using the TRIZOL reagent protocol. Genomic DNA was then digested by methylation-sensitive restriction enzymes, amplified by polymerase chain reaction (PCR), electrophoresed on a 10% polyacrylamide gel and stained using SYBR Gold. Real-time PCR was also performed to verify the transcript levels.
The CpG dinucleotides analysed were observed to be unmethylated in the majority of DNA samples of the three groups and statistical differences were not found among groups (p>0.05). However, a trend towards methylation was observed in the TLR2 HhaI site in the samples of the periodontitis non-smoker groups. In fact, the analysis of all CpG sites together shows which complete methylation is observed in the shortest level in the samples of periodontitis non-smoker group. The analysis of transcript levels demonstrated no difference among groups (p>0.05).
The results demonstrated major unmethylation of the TLR4 gene promoter in all groups. However, the results for the TLR2 gene promoter are inconclusive; this gene was found as a mosaic of methylated and unmethylated DNA in the majority of samples of the three groups and we also observed a trend towards the DNA methylation of CpG sites recognized by the HhaI enzyme.
本研究旨在分析健康受试者、吸烟者和非吸烟者慢性牙周炎患者牙龈组织样本中 toll 样受体（TLR）2 和 TLR4 基因启动子区域 DNA 甲基化的状态。
使用 TRIZOL 试剂方案从牙龈组织中提取基因组 DNA 和总 RNA。然后使用甲基敏感限制性内切酶消化基因组 DNA，通过聚合酶链反应（PCR）扩增，在 10％聚丙烯酰胺凝胶上电泳，并用 SYBR Gold 染色。还进行了实时 PCR 以验证转录水平。
分析的 CpG 二核苷酸在三组大多数 DNA 样本中观察为未甲基化，组间无统计学差异（p>0.05）。然而，在非吸烟者牙周炎组的 TLR2 HhaI 位点观察到甲基化趋势。事实上，对所有 CpG 位点的分析表明，非吸烟者牙周炎组样本中观察到最短水平的完全甲基化。转录水平分析表明组间无差异（p>0.05）。
结果表明 TLR4 基因启动子在所有组中主要是非甲基化的。然而，TLR2 基因启动子的结果尚无定论；该基因在三组大多数样本中被发现为甲基化和非甲基化 DNA 的嵌合体，我们还观察到 HhaI 酶识别的 CpG 位点的 DNA 甲基化趋势。
