抗肿瘤坏死因子单抗对脂多糖诱导的人牙周膜成纤维细胞TRAIL和TNF-α的影响

摘要/Abstract

摘要： 目的: 观察脂多糖对人牙周膜成纤维细胞(HPLFs )肿瘤坏死因子(TNF)受体相关凋亡诱导配体(TRAIL) 和TNF-α的诱导作用及抗肿瘤坏死因子单抗(抗TNF单抗)对其的影响,探讨TNF超家族参与牙周病的可能作用机制。方法: HPLFs培养至第6代, 加入不同浓度的脂多糖(0、0.1、1、10、100 μg/mL)培养 24 h,分为命名为Z₀组、Z_0.1组、Z₁组、Z₁₀组、Z₁₀₀组,并取Z₁组浓度的脂多糖,在加药的同时加抗TNF单抗 75 μg/mL( Z₁+75组)。分别采用实时荧光定量PCR法或Western blot法测定TRAIL 和TNF-α mRNA或蛋白的表达。结果: Z₁组、Z₁₀组HPLFs TRAIL mRNA的表达水平显著高于Z₀组或Z_0.1 组(均P<0.05),Z₁组与Z₁₀组之间差异无统计学意义(P>0.05); Z₁₀₀组显著高于Z₀组(P<0.05)且与Z_0.1组、Z₁组和Z₁₀组比较差异无统计学意义(均P>0.05)。Z₁组或Z₁₀组TNF-α mRNA的表达水平显著高于Z₀组或Z₁₀₀组(均P<0.05);Z₁组与Z₁₀组之间,Z₀组、Z_0.1组和Z₁₀₀组之间差异无统计学意义(均P>0.05)。HPLFs TRAIL 蛋白的表达水平在Z₁₀₀组<Z₀组<Z_0.1组<Z₁组(均P<0.05 或<0.01);Z₁₀组显著高于Z₀组或Z₁₀₀组(均P<0.01),但与Z_0.1组或Z₁组之间差异无统计学意义(均P>0.05)。抗TNF单抗治疗后TRAIL mRNA及蛋白和TNF-α mRNA的表达水平显著低于Z₁组(P<0.05 和P<0.01)。TRAIL和TNF-α mRNA的表达水平呈显著直线正相关(r=0.819,n=30, P<0.01)。结论: HPLFs经脂多糖诱导后,其TRAIL和TNF-α的表达呈现先升高后下降,且随脂多糖的浓度变化而相应地变化,这种诱导作用能被抗TNF单抗所抑制。

关键词: 人牙周膜成纤维细胞, 肿瘤坏死因子, TRAIL, 脂多糖, 抗肿瘤坏死因子单抗

Abstract: AIM: To investigate the possible roles of tumor necrosis factor(TNF) superfamily in the pathogenesis of periodontal disease inflammation, the effect of anti-tumor necrosis factor antibody on TRAIL and TNF-α expression induced by lipopolysaccharide in human periodontal ligament fibroblasts is observed.METHODS: HPLFs were cultured to the sixth generation cells and were interfered with defferent concentrations of lipopolysaccharide(0,0.1,1,10,100 μg/mL,respectively) for 24 h, named group Z₀,Z_0.1,Z₁,Z₁₀ and Z₁₀₀. HPLFs were also cultured with 1 μg/mL lipopolysaccharide and 75 μg/mL anti- TNF antibody (named group Z₁+75). The expressions of both TNF-α and TRAIL mRNA were analysed by Real-time quantitative reverse transcription polymerase chain reaction(QRT-PCR),while TRAIL protein were determined by West Blot methods.RESULTS: The expressions of TRAIL mRNA in group Z₁ or Z₁₀ were significantly higher than those in group Z₀ or Z_0.1 (all P<0.05),in group Z₁₀₀ were significantly higher than those in group Z₀(P<0.05).But there were no significant differences neither between group Z₁ and Z₁₀ nor among group Z₁₀₀, Z_0.1, Z₁ and Z₁₀ (all P>0.05). Moreover, the expressions of TNF-α mRNA in group Z₁ or Z₁₀ were significantly higher than those in group Z₀ or Z₁₀₀ (all P<0.05), whereas there were nonsignificant differences neither between group Z₁ and Z₁₀ nor among group Z₀, Z_0.1, and Z₁₀₀ (all P>0.05). Dramatically, the expressions of TRAIL protein were significant difference between each groups, in group Z₁₀₀ <Z₀ < Z_0.1 <Z₁(all P<0.05 or P<0.01).And it was significantly higher in group Z₁₀ than those in group Z₀ or Z₁₀₀(all P<0.01),while there were nonsignificant differences neither between group Z₁₀ and Z_0.1 nor among group Z₁₀ and Z₁ (all P>0.05). Noteworthy, the expressions of either TRAIL mRNA and protein or TNF-α mRNA in group Z₁+75 were significantly decreased than those in group Z₁(P<0.05 or <0.01,respectively). Furthermore,there were significance positive correlation between levels of TRAIL and TNF-α mRNA(r=0.819,n=30, P<0.01).CONCLUSION: The results shows that the expressions of TRAIL and TNF-α in HPLFs can be induced by lipopolysaccharide, the levels of them were elevated in the first and decreased subsequently, this changes were accompanied by lipopolysaccharide concentration. Date also show the function of lipopolysaccharide can be inhibited by anti-TNF antibody.

Key words: human periodontal ligament fibroblasts, tumor necrosis factor, TNF-related apoptosis-inducing ligand, lipopolysaccharide, anti-tumor necrosis factor antibody, periodontal disease

中图分类号:

陈焱, 叶斌, 莫文元, 俞诚波, 林晡, 尹向鹏, 许红苗. 抗肿瘤坏死因子单抗对脂多糖诱导的人牙周膜成纤维细胞TRAIL和TNF-α的影响[J]. 中国临床药理学与治疗学, 2015, 20(5): 493-498.

CHEN Yan, YE Bin, MOU Wen-yuan, YU Cheng-bo, LIN Bu, YIN Xiang-peng, XU Hong-miao. Effect of anti-tumor necrosis factor antibody on TRAIL and TNF-α expression induced by lipopolysaccharide in human periodontal ligament fibroblasts[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2015, 20(5): 493-498.

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