肿瘤坏死因子α可激活NF-κB信号通路抑制牙周膜干细胞成骨分化
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摘要
背景:既往研究大多侧重于炎症因子激活NF-κB信号通路对骨细胞的影响,关于微炎症环境对人牙周膜干细胞成骨分化及NF-κB信号通路的影响研究较少。目的:探讨肿瘤坏死因子α对人牙周膜干细胞骨向分化及NF-κB信号通路的影响。方法:通过酶消化结合组织块法体外培养人牙周膜干细胞,免疫组织化学染色鉴定人牙周膜干细胞来源。该研究获得青海大学附属医院伦理委员会批准,患者均签署知情同意书。将人牙周膜干细胞分为2组,对照组用单纯成骨诱导液培养,肿瘤坏死因子α组用单纯成骨诱导液+10μg/L肿瘤坏死因子α培养。在成骨诱导不同时间点,进行茜素红染色定量分析、碱性磷酸酶活性检测,实时定量RT-PCR检测Osterix、Runx2基因表达水平,Western Blot检测NF-κB信号通路关键蛋白表达水平。结果与结论:①免疫组织化学染色结果显示人牙周膜干细胞抗波形丝蛋白染色表达阳性,抗角蛋白表达阴性;②茜素红染色后肿瘤坏死因子α组形成的矿化结节比对照组数量少、范围小、染色浅;③肿瘤坏死因子α组碱性磷酸酶活性明显低于对照组(P <0.05);④肿瘤坏死因子α组Osterix、Runx2基因表达水平均显著低于对照组(P <0.05);⑤肿瘤坏死因子α组p-IκBα蛋白表达量显著高于对照组(P <0.05),p65、IκBα蛋白表达量显著低于对照组(P<0.05);⑥结果表明,肿瘤坏死因子α能够通过激活NF-κB信号通路抑制人牙周膜干细胞成骨分化。
BACKGROUND: Previous studies have focused on the effects of inflammatory factors-activated nuclear factor-kappa B signaling pathway on bone cells. Little has been reported on the effects of micro-inflammatory environment on osteogenic differentiation and nuclear factor-kappa B signaling pathways of human periodontal ligament stem cells.OBJECTIVE: To investigate the effect of tumor necrosis factor-alpha on osteogenic differentiation and nuclear factor-kappa B signaling pathway of periodontal ligament stem cells.METHODS: Human periodontal ligament stem cells were cultured in vitro by enzymatic digestion combined with tissue explant method, and the cell source was identified by immunohistochemical staining. The study protocol was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University, and informed consent was obtained from each patient prior to the enrollment in the study. The cells were cultured in simple osteogenic induction medium(control group) or osteogenic induction medium containing 10 μg/L tumor necrosis factor-alpha(tumor necrosis factor-alpha group). Alizarin red staining and alkaline phosphatase activity were used to detect the effect of tumor necrosis factor-alpha on osteogenic differentiation of periodontal ligament stem cells. Real-time quantitative RT-PCR was used to detect the effect of tumor necrosis factor-alpha on the expression of osteogenic differentiation genes, Osterix and Runx2. Western blot was used to detect the effect of tumor necrosis factor-alpha on nuclear factor-kappa B signaling pathway.RESULTS AND CONCLUSION: Immunohistochemical staining showed that human periodontal ligament stem cells were positive for anti-vimentin and negative for anti-keratin. After alizarin red staining, mineralized nodules formed in the tumor necrosis factor-alpha group were fewer in number, smaller in scope and lighter in staining than those in the control group. Alkaline phosphatase activity in the tumor necrosis factor-alpha group was significantly lower than that in the control group(P < 0.05). Real-time quantitative RT-PCR results showed that the expression levels of Osterix and Runx2 mRNA in tumor necrosis factor-alpha group were significantly lower than those in the control group(P < 0.05). Western blot assay showed that, compared with the control group, the expression of p-IκBα protein increased significantly in the tumor necrosis factor-alpha group(P < 0.05), while the expression of p65 and IκBα proteins decreased(P < 0.05). These findings indicate that tumor necrosis factor-alpha can inhibit the osteogenic differentiation of human periodontal ligament stem cells by activating the nuclear factor-kappa B signaling pathway.
BACKGROUND: Previous studies have focused on the effects of inflammatory factors-activated nuclear factor-kappa B signaling pathway on bone cells. Little has been reported on the effects of micro-inflammatory environment on osteogenic differentiation and nuclear factor-kappa B signaling pathways of human periodontal ligament stem cells.OBJECTIVE: To investigate the effect of tumor necrosis factor-alpha on osteogenic differentiation and nuclear factor-kappa B signaling pathway of periodontal ligament stem cells.METHODS: Human periodontal ligament stem cells were cultured in vitro by enzymatic digestion combined with tissue explant method, and the cell source was identified by immunohistochemical staining. The study protocol was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University, and informed consent was obtained from each patient prior to the enrollment in the study. The cells were cultured in simple osteogenic induction medium(control group) or osteogenic induction medium containing 10 μg/L tumor necrosis factor-alpha(tumor necrosis factor-alpha group). Alizarin red staining and alkaline phosphatase activity were used to detect the effect of tumor necrosis factor-alpha on osteogenic differentiation of periodontal ligament stem cells. Real-time quantitative RT-PCR was used to detect the effect of tumor necrosis factor-alpha on the expression of osteogenic differentiation genes, Osterix and Runx2. Western blot was used to detect the effect of tumor necrosis factor-alpha on nuclear factor-kappa B signaling pathway.RESULTS AND CONCLUSION: Immunohistochemical staining showed that human periodontal ligament stem cells were positive for anti-vimentin and negative for anti-keratin. After alizarin red staining, mineralized nodules formed in the tumor necrosis factor-alpha group were fewer in number, smaller in scope and lighter in staining than those in the control group. Alkaline phosphatase activity in the tumor necrosis factor-alpha group was significantly lower than that in the control group(P < 0.05). Real-time quantitative RT-PCR results showed that the expression levels of Osterix and Runx2 mRNA in tumor necrosis factor-alpha group were significantly lower than those in the control group(P < 0.05). Western blot assay showed that, compared with the control group, the expression of p-IκBα protein increased significantly in the tumor necrosis factor-alpha group(P < 0.05), while the expression of p65 and IκBα proteins decreased(P < 0.05). These findings indicate that tumor necrosis factor-alpha can inhibit the osteogenic differentiation of human periodontal ligament stem cells by activating the nuclear factor-kappa B signaling pathway.
引文
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[23] Yeh CJ, Lin PY, Liao MH, et al. TNF-alpha mediates pseudorabies virus-induced apoptosis via the activation of p38 MAPK and JNK/SAPK signaling. Virology. 2008;381(1):55-66.
[24]于钦,陈贤.牙周炎患者基础治疗前后唾液和龈沟液中IL-6、TNF-α、MMP-8水平的变化[J].北京口腔医学, 2018,26(6):336-339.
[25]于莉,李淑慧,袁萍,等. TNF-α对根尖乳头干细胞体外增殖及成牙成骨向分化能力的影响[J].口腔医学研究,2016, 32(4):343-346.
[26]王光.雌激素缺乏导致的骨质疏松环境中TNF-α通过MiR-21影响小鼠骨髓间充质干细胞成骨分化能力的研究[D].西安:第四军医大学, 2012.
[27]曹蓉蓉,徐江,李淑慧,等. TNF-α影响鼠根尖乳头干细胞成骨/成牙本质能力的实验研究[J].口腔医学, 2017,37(3):208-213.
[28]王有为.炎性因子TNF-α对SD大鼠骨髓间充质干细胞成骨分化的影响[D].沈阳:中国医科大学,2015.
[29]张冬梅,刘静波,潘亚萍.牙龈卟啉单胞菌感染血管内皮细胞对NF-κB和p38MAPK信号通路的影响[J].中国医科大学学报, 2011,40(6):530-533.
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[31] Egbuniwe O, Grover S, Duggal AK, et al. TRPA1 and TRPV4 activation in human odontoblasts stimulates ATP release. J Dent Res. 2014;93(9):911-917.
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[2]潘有条,王一飞,赵洵.微环境对牙周膜干细胞分化的抑制和诱导作用[J].国际口腔医学杂志,2016,43(2):207-211.
[3]刘娜,李华,张洋,等.炎症微环境作用下非经典Wnt/Ca2+信号通路对牙周膜干细胞成骨分化的影响[J].中华老年口腔医学杂志, 2015,13(5):257-262.
[4]聂嘉,张博,顾斌,等.p38丝裂原活化蛋白激酶在炎症微环境作用下对牙周膜干细胞成骨分化的影响[J].中国医学科学院学报, 2015, 37(1):1-7.
[5]冯娟,刘亚蕊,张清彬,等.炎性微环境对牙周膜干细胞骨向分化作用的初步研究[J].医学信息, 2015,28(26):23.
[6]杨昊.炎症刺激对人牙龈间充质干细胞和牙周膜干细胞生物学性能的影响[D].西安:第四军医大学, 2013.
[7]杨芳,陈明月,胡英英,等.PPARγ在脂多糖刺激牙周膜细胞中调控NF-κB信号通路的作用研究[J].口腔医学研究, 2017, 33(7):698-702.
[8]王月昊,王小琴,苗伟,等.间歇性低氧对牙周炎大鼠牙周组织中NF-κB、IL-6及PGE2含量的影响[J].实用口腔医学杂志, 2016,32(1):28-31.
[9]陈铁楼,苏丹,许兵.NF-κB分子生物学特性及其在牙周炎等炎症性疾病中的作用[J].口腔医学,2010,30(4):242-245.
[10] Zhang J, Li ZG, Si YM, et al. The difference on the osteogenic differentiation between periodontal ligament stem cells and bone marrow mesenchymal stem cells under inflammatory microenviroments. Differentiation. 2014;88(4-5):97-105.
[11] Chen X, Hu C, Wang G, et al. Nuclear factor-κB modulates osteogenesis of periodontal ligament stem cells through competition withβ-catenin signaling in inflammatory microenvironments. Cell Death Dis. 2013;4:e510.
[12]和璐,廖雁婷.牙周炎与2型糖尿病[J].中华糖尿病杂志, 2010,02(4):242-245.
[13]袁萍,李淑慧,赵璐,等.炎症微环境下人牙周膜干细胞的生物学特性[J].中国组织工程研究, 2016, 20(6):898-905.
[14]徐洁.慢性牙周炎与2型糖尿病的相互关系及影响因素分析[J].航空航天医学杂志, 2015,26(2):146-148.
[15]吴丹,程功,李晶,等.牙菌斑培养菌群宏基因组文库构建及抗生素耐药基因筛选[J].微生物学通报, 2013, 40(6):1041-1048.
[16]张欣然,刘翠,朱彪,等.肿瘤坏死因子-α诱导骨髓间充质干细胞凋亡作用的研究[J].牙体牙髓牙周病学杂志, 2015,25(7):391-395.
[17]翟启明,李蓓,刘露,等.线粒体融合蛋白-1对牙周膜干细胞成骨分化能力的影响[J].实用口腔医学杂志, 2018,34(2):172-177.
[18]杨柳青,徐雪,王黎明,等.慢性牙周炎患者龈沟液中IL-8和TNF-α水平变化及临床意义[J].现代生物医学进展, 2016, 16(30):5933-5936.
[19]薛冬梅,欧阳燕.尼美舒利对慢性牙周炎患者疗效及龈沟液中IL-8和TNF-α水平的影响[J].中国生化药物杂志, 2016,36(4):77-79.
[20]李晓光,王一珠,郭斌.慢性牙周炎中肿瘤坏死因子α对骨髓间充质干细胞成骨分化的调控作用[J].华西口腔医学杂志, 2017,15(3):114-118.
[21]王琳源,靳赢,林晓萍.适应性免疫应答在牙周炎发生中的作用[J].中华口腔医学杂志,2013,48(2):115-118.
[22]朱治宇,刘国勤.慢性牙周炎治疗前后患牙龈沟液中IL-8和TNF-α水平变化比较[J].细胞与分子免疫学杂志,2010,26(11):156-159.
[23] Yeh CJ, Lin PY, Liao MH, et al. TNF-alpha mediates pseudorabies virus-induced apoptosis via the activation of p38 MAPK and JNK/SAPK signaling. Virology. 2008;381(1):55-66.
[24]于钦,陈贤.牙周炎患者基础治疗前后唾液和龈沟液中IL-6、TNF-α、MMP-8水平的变化[J].北京口腔医学, 2018,26(6):336-339.
[25]于莉,李淑慧,袁萍,等. TNF-α对根尖乳头干细胞体外增殖及成牙成骨向分化能力的影响[J].口腔医学研究,2016, 32(4):343-346.
[26]王光.雌激素缺乏导致的骨质疏松环境中TNF-α通过MiR-21影响小鼠骨髓间充质干细胞成骨分化能力的研究[D].西安:第四军医大学, 2012.
[27]曹蓉蓉,徐江,李淑慧,等. TNF-α影响鼠根尖乳头干细胞成骨/成牙本质能力的实验研究[J].口腔医学, 2017,37(3):208-213.
[28]王有为.炎性因子TNF-α对SD大鼠骨髓间充质干细胞成骨分化的影响[D].沈阳:中国医科大学,2015.
[29]张冬梅,刘静波,潘亚萍.牙龈卟啉单胞菌感染血管内皮细胞对NF-κB和p38MAPK信号通路的影响[J].中国医科大学学报, 2011,40(6):530-533.
[30] Lin G, Chen S, Lei L, et al. Effects of Intravenous Injection of Porphyromonas gingivalis on Rabbit Inflammatory Immune Response and Atherosclerosis. Mediators Inflamm. 2015;2015:364391.
[31] Egbuniwe O, Grover S, Duggal AK, et al. TRPA1 and TRPV4 activation in human odontoblasts stimulates ATP release. J Dent Res. 2014;93(9):911-917.
[32]刘亚丽,刘文佳,胡成虎,等.牙周慢性炎症对牙周膜干细胞生物学特性的影响[J].牙体牙髓牙周病学杂志,2014,24(1):21-25.
[33]王愉惠.炎症微环境下牙周膜干细胞成骨分化的调控机制[J].临床口腔医学杂志, 2017,33(7):441-444.
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