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

Effect of anti-tumor necrosis factor antibody on TRAIL and TNF-α expression induced by lipopolysaccharide in human periodontal ligament fibroblasts

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、100μg/m L)培养24 h,分为命名为Z0组、Z0.1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75μg/m L(Z1+75组)。分别采用实时荧光定量PCR法或Western blot法测定TRAIL和TNF-αm RNA或蛋白的表达。结果:Z1组、Z10组HPLFs TRAIL m RNA的表达水平显著高于Z0组或Z0.1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Z0.1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-αm RNA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、Z0.1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<Z0.1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Z0.1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 m RNA及蛋白和TNF-αm RNA的表达水平显著低于Z1组(P<0.05和P<0.01)。TRAIL和TNF-αm RNA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。

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 /m L,respectively） for 24 h,named group Z0,Z0. 1,Z1,Z10 and Z100. HPLFs were also cultured with 1 μg / m L lipopolysaccharide and75 μg / m L anti- TNF antibody（ named group Z1＋75）. The expressions of both TNF-α and TRAIL m RNA were analysed by Real-time quantitative reverse transcription polymerase chain reaction（ QRTPCR）,while TRAIL protein were determined by West Blot methods. RESULTS： The expressions of TRAIL m RNA in group Z1 or Z10were significantly higher than those in group Z0 or Z0. 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 Z10nor among group Z100,Z0. 1,Z1 and Z10（all P〈0. 05）.Moreover,the expressions of TNF-α m RNA in group Z1 or Z10were significantly higher than those in group Z0 or Z100（all P〈0. 05）,whereas there were nonsignificant differences neither between group Z1 and Z10nor among group Z0,Z0. 1,and Z100（all P〈0. 05）. Dramatically,the expressions of TRAIL protein were significant difference between each groups,in group Z100 Z0 Z0. 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 Z0. 1nor among group Z10 and Z1（all P〈0. 05）. Noteworthy, the expressions of either TRAIL m RNA and protein or TNF-α m RNA 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 m RNA（ 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.