曲古抑菌素A对肿瘤坏死因子α诱导的炎症微环境下牙周膜干细胞成骨分化能力的影响

Effect of trichostatin A on the osteogenic differentiation potential of periodontal ligament stem cells in inflammatory microenvironment induced by tumor necrosis factor-α stimulation

目的 观察肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）诱导的炎症微环境下牙周膜干细胞（periodontal ligament stem cells,PDLSC）中组蛋白去乙酰化酶（histone deacetylases,HDAC）家族的表达变化,探究HDAC抑制剂曲古抑菌素A（trichostatin A,TSA）干扰HDAC功能后对PDLSC成骨分化能力的影响.方法 收集新鲜健康的离体牙牙周膜组织,分离培养PDLSC.采用实时定量PCR技术检测对照组及TNF-α（10 μg/L）刺激下HDAC1～11的表达变化;甲基噻唑基四唑（methyl thiazolyl tetrazolium,MTT）法检测TSA（50 nmol/L）对PDLSC增殖能力的影响,对照组加正常细胞培养液,TNF-α组加含TNF-α的细胞培养液,TSA组加含TNF-α及TSA的细胞培养液;分别采用茜素红染色、实时定量PCR技术和蛋白质印迹法检测TSA对炎症微环境下PDLSC成骨分化能力的影响,对照组加正常成骨诱导液,TNF-α组加含TNF-α的成骨诱导液,TSA组加含TNF-α及TSA的成骨诱导液.结果 除HDAC7外,TNF-α组HDAC的表达均显著高于对照组（P＜0.05）;50 nmol/L TSA对PDLSC的增殖能力无显著影响（P=0.232）;茜素红染色显示TNF-α组PDLSC较对照组基质矿化显著减少,而TSA组细胞矿化能力明显提高;与对照组（设为1.0）相比,TNF-α组PDLSC成骨相关基因核心结合蛋白因子2（runt-related transcription factor-2,RUNX2）、碱性磷酸酶（alkaline phosphatase,ALP） mRNA的相对表达量分别为0.17±0.02、0.32±0.03,TSA组RUNX2、ALP mRNA的表达量（分别为0.67±0.03、0.89±0.02）均显著高于TNF-α组（P＜0.01）;蛋白质印迹法结果显示,TNF-α组PDLSC成骨相关蛋白的表达较对照组明显减少,与TNF-α.组相比,TSA可以明显促进炎症微环境下细胞成骨相关蛋白的表达.结论 炎症微环境下PDLSC高表达HDAC,应用TSA抑制HDAC后可显著提高炎症环境下PDLSC的成骨分化能力.

Objective To compare the expression of histone deacetylase（HDAC）1-11 of humanperiodontal ligament stem cells（PDLSC） in normal and inflammatory microenvironments,and to investigate the effect of histone deacetylase inhibitor trichostatin A（TSA） on the osteogenic differentiation potential of PDLSC in inflammatory microenvironment induced by tumor necrosis factor-α（TNF-α） stimulation.Methods PDLSC were isolated from periodontal ligament tissues obtained from the surgically extracted human teeth and cultured by single-colony selection.The expression of HDAC1-11 in cells with or without TNF-α（10 μg/L） stimulation was evaluated by quantitative real time-PCR（RT-PCR）.The effect of TSA on cell proliferation was investigated by methyl thiazolyl tetrazolium（MTT） assay.The influence of TSA on osteogenic differentiation of PDLSC in inflammatory microenvironment with TNF-α stimulation was assessed by alizarin red staining,qoantitative RT-PCR and Western blotting,respectively.Results The expression of HDAC in PDLSC with TNF-α stimulation was significantly higher than that in normal PDLSC（P〈0.05） （except HDAC7,P=0.243）.TSA had no significant effect on PDLSC proliferation at the concentration of 50 nmol/L（P=0.232）.The alizarin red staining showed that PDLSC in TNF-α group generated less mineralized nodule than the control group,while the cell matrix mineralization in TSA group was improved obviously.TNF-α had an inhibitory effect on the expression of osteogenesis related genes,runt-related transcription factor-2（RUNX2） and alkaline phosphatase（ALP）,with relative gene expression ratio （experimental/control） decreased to 0.17 ±0.02 and 0.32±0.03,while TSA could significantly increase the genes＇ expression to 0.67±0.03 and 0.89±0.02（P〈0.01）.Western blotting test showed that in TNF-α group the expression of osteogenesis related proteins was obviously reduced,and compared with the TNF-α group,TSA could significantly promote the expression of proteinsin inflammatory microenvironment.Conclusions PDLSC in inflammatory microenvironment by TNF-α stimulation had a higher expression of HDAC than that in normal conditions.TSA,as a histone deacetylase inhibitor,could significantly promote the osteogenic differentiation potential of PDLSC in inflammatory microenvironment by suppressing HDAC.