高糖对牙龈卟啉单胞菌脂多糖刺激人成纤维细胞分泌炎症因子的影响

Effect of high glucose on the secretion of cytokines induced by Porphyromonas gingivalis lipopolysaccharide

目的 探讨高浓度葡萄糖对牙龈卟啉单胞菌（Porphyromonas gingivalis,Pg）脂多糖刺激人牙龈成纤维细胞（human gingival fibroblasts,HGF）分泌炎症细胞因子的影响及作用机制,进一步探讨糖尿病与牙周炎的相互关系.方法 组织块法培养HGF,细胞处理分为以下4组：A组：低糖（5.5 mmol/L葡萄糖）＋1 mg/L Pg脂多糖刺激组;B组：低糖＋10 mg/L Pg脂多糖刺激组;C组：高糖（25 mmol/L葡萄糖）＋1 mg/L Pg脂多糖刺激组;D组：高糖＋10 mg/L Pg脂多糖刺激组.采用酶联免疫法检测4组HGF细胞6、12h后分泌肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）和白细胞介素1β（interleukin-1β,IL-1β）的变化,实时定量PCR检测Toll样受体（toll-like receptor,TLR）2和TLR4在HGF中的表达.在高糖＋10 mg/L Pg脂多糖组采用抗TLR2、抗TLR4单克隆抗体预先处理HGF,检测HGF中12h的TNF-α、IL-1β水平,以高糖＋10 mg/L Pg脂多糖刺激组为对照组,并进行统计学分析.结果 相同质量浓度的Pg脂多糖刺激HGF 6、12h后高糖条件（C组、D组）下TNF-α及IL-1β分泌及TLR2 mRNA的表达均较低糖条件下（A组、B组）显著升高（P值均＜0.01）,TLR4 mRNA的表达C组与A组相比表达升高,但差异无统计学意义（P＞0.05）;D组显著高于B组（P＜0.01）.高糖＋10 mg/L Pg脂多糖刺激组中分别阻断TLR2、4后,TNF-α的分泌量分别为（297.16± 11.49）、（390.01±12.81） ng/L,均显著低于对照组[（459.80±8.04） ng/L]（F=166.02,P＜0.01）,IL-1β的分泌量分别为（49.90±4.08）、（99.35±5.01） ng/L,亦均显著低于对照组[（147.37±9.87） ng/L]（F=153.51,P＜0.000 1）.结论 高糖可明显促进Pg脂多糖刺激HGF后炎症细胞因子的分泌,其作用可能通过调节HGF表面TLR2和TLR4的表达来实现,在一定程度上证实糖尿病患者血糖升高可加重牙周炎症.

Objective To investigate the influence of high glucose on Porphyromonasgingivalis（Pg）lipopolysaccharide（LPS） stimulating human gingival fibroblasts（HGF） to secret the cytokines.Methods HGF were obtained from the primary culture of the tissue explants.Cells were divided into four groups,low glucose（5.5 mmol/L） ＋ 1 mg/L Pg LPS（group A）; low glucose＋ 10 mg/L Pg LPS（group B）; high glucose （25 mmol/L） ＋ 1 mg/L Pg LPS（group C）;high glucose＋ 10 mg/L Pg LPS（group D）.The levels of tumor necrosis factor-α（TNF-α） and interleukin-1β（IL-1β） in cell supernatants were detected by enzyme-linked immunosorbent assay at 6 h and 12 h.The expressions of toll-like receptor 2,4（TLR-2,4） were examined by real-time polymerase chain reaction.After pretreatment with anti-TLR2 and anti-TLR4 monoclonal antibody in HGF,TNF-α and L-1β levels were detected.Results TNF-α concentration increased obviously in high glucose 6 h and 12 h after Pg LPS stimulation（P〈0.01）.IL-1β secretion increased（P〈0.01）.Meanwhile,TLR2,4 mRNA expression increased,especially in high glucose＋ 10 mg/L Pg LPS（P〈0.01）.After inhibition of the TLR2,4 in high glucose＋ 10 mg/L Pg LPS respectively,TNF-α level[（297.16± 11.49）,（390.01 ± 12.81） ng/L] decreased（F=166.02,P〈0.01）,and IL-1β level[（49.90±4.08）,（99.35±5.01） ng/L] also decreased （F=153.51,P〈0.01）.Conclusions High glucose may promote Pg LPS to stimulate the secretion of TNF-α and IL-1β through regulating TLR2,4 expression,which suggests that the elevating blood glucose precipitate in aggravating the process of periodontal disease.