TRAF6敲低对低氧状态下IL-17调控人牙周膜成纤维细胞表达OPG及RANKL的影响

Effects of TRAF6 knockdown on the IL-17 modulating the expression of OPG and RANKL in periodontal ligament cells under hypoxia

目的:探讨敲低肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)对人牙周膜成纤维细胞(human periodontal ligament cells,h PDLCs)在低氧状态经白介素17(interleukin 17,IL-17)刺激表达骨保护素(osteoprotegerin,OPG)及核因子κB受体活化因子配体(receptor activator for nuclear factor-κB ligand,RANKL)的影响。方法:将h PDLCs细胞设为常氧加IL-17组、低氧加IL-17组以及单纯低氧组,每组内分为阴性对照组(TRAF6-NC组)和干扰组(TRAF6-sh RNA组)。将靶向TRAF6基因的sh RNA慢病毒载体(TRAF6-sh RNA)感染h PDLCs,通过倒置显微镜观察病毒感染效率并用RT-PCR方法验证,用Western blot及RT-PCR方法检测分析h PDLCs低氧诱导因子(hypoxia inducible factor-1,HIF-1)、OPG及RANKL的表达变化。结果:低氧加IL-17组RANKL与OPG蛋白相对表达量的比值RANKL/OPG高于常氧加IL-17组及低氧组。在低氧加IL-17条件下,与TRAF6-NC组相比,TRAF6-sh RNA组RANKL表达减少,RANKL与OPG相对表达量的比值RANKL/OPG减少。结论:TRAF6参与低氧状态下IL-17刺激h PDLCs表达骨吸收分子的调控,敲低TRAF6可使低氧状态下IL-17诱导的h PDLCs表达骨吸收分子减少。

Objective：This study aimed at the effect of tumor necrosis factor receptor-associated factor 6（TRAF6）knockdown oninterleukin-17（IL-17）modulating the expression of osteoprotegerin（OPG）and receptor activator for nuclear factor-κB ligand（RANKL）in human periodontal ligament cells（h PDLCs）under hypoxia. Methods：The experiment was set as the normoxia plus IL-17 group,thehypoxia plus IL-17 group and the hypoxia group. Each group was divided into the negative control group（the TRAF6-NC group）andthe disturbance group（the TRAF6-sh RNA group）. The sh RNA lentiviral vector was used to infect h PDLCs. The efficiency of infectionwas observed by inverted microscope and verified by RT-PCR. The expression of HIF-1α,RANKL and OPG in human periodontalligament cells were detected by Western blot and RT-PCR. Results：The ratio of RANKL/OPG relative expression of RANKL and OPGprotein in the hypoxia plus IL-17 group was higher than that in the hypoxia group. Under hypoxia plus IL-17 conditions,the expressionof RANKL was decreased in the TRAF6-sh RNA group compared with the TRAF6-NC group,and the ratio of RANKL/OPG relativeexpression of RANKL and OPG was decreased. Conclusion：TRAF6 is involved in the regulation of IL-17-stimulated expression ofbone resorption molecules in human periodontal ligament cells in hypoxic conditions. Knocking down TRAF6 can reduce IL-17-induced expression of bone resorption molecules in human periodontal ligament cells.