TRAF6沉默对LPS刺激牙周膜成纤维细胞成骨分化的影响

Effect of TRAF6 Knockdown on Osteogenic Differentiation of Human Periodontal Ligament Cell Treated with LPS

目的:使用小干扰RNA(small interfering RNA,siRNA)沉默人牙周膜成纤维细胞(human periodontal ligament cell,hPDLC)肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor associated factor 6,TRAF6)的基因,观察沉默TRAF6基因对LPS刺激的hPDLC成骨分化的影响。方法:实验分为空白对照组、转染试剂组、TRAF6siRNA组、control siRNA组,采用脂质体法将TRAF6siRNA、control siRNA分别瞬时转染入对应组中,设转染试剂组加入等量的转染试剂,空白对照组不做处理,再使用10 mg/L LPS刺激细胞,RT-PCR、Western blot检测转染后TRAF6的基因及蛋白表达水平,使用LPS+成骨诱导液培养细胞,碱性磷酸酶检测试剂盒检测碱性磷酸酶的活性,RT-PCR检测Runx-2和I型胶原蛋白(Col-I)基因表达情况。结果:TRAF6siRNA组的TRAF6mRNA和蛋白的表达显著降低(P<0.001);成骨诱导3d后,LPS刺激下各组ALP的表达量要显著低于对照组(P<0.05),TRAF6siRNA组的ALP表达量要高于空白对照、转染试剂组和control siRNA组(P<0.05),TRAF6siRNA的Runx-2和Col-I的mRNA表达均明显高于其余3组(P<0.05)。结论:沉默TRAF6基因能减轻LPS对hPDLC成骨分化的抑制作用,即沉默TRAF6基因能促进LPS刺激下的hPDLC成骨分化,推测TRAF6可能影响牙周炎的发生发展进程,是牙周炎潜在的治疗靶点。

Objective：To observe the effect of tumor necrosis factor receptor-related factor 6（TRAF6）knockdown on osteogenic differentiation of human periodontal ligament cell（hPDLC）stimulated by LPS.Methods：The TRAF6 siRNA and control siRNA were transiently transfected into hPDLC.The cells were stimulated with 10μg/ml LPS.The silence efficiency of TRAF6 was detected by RT-PCR and Western blot.The expression of ALP was detected by alkaline phosphatase assay kit.The expression of Runx-2 and type I collagen（Col-I）gene was detected by RT-PCR.Results：The expression of TRAF6 mRNA and protein in TRAF6 siRNA group was significantly decreased（P0.001）.The expression of ALP in LPS-stimulated group was significantly lower than that in nonLPS-stimulated group（P0.05）.The expression of ALP,Runx-2,and Col-I mRNA in TRAF6 siRNA was significantly higher than that in the other three groups（P0.05）.Conclusion：TRAF6 knockdown can alleviate the inhibitory effect of LPS on osteogenesis differentiation of hPDLC,which indicates that TRAF6 may affect the development of periodontitis and may be a potential therapeutic target for periodontitis.