施琼玲,吴婷婷,李静,汪艳,黄慧.TNF-a对BMP-2诱导下小鼠BMSCs成骨分化miRNA表达的影响[J].口腔材料器械杂志,2014,23(3):130-136.
TNF-a对BMP-2诱导下小鼠BMSCs成骨分化miRNA表达的影响
Effect of TNF-αon the miRNA expression profile in osteogenic differentiation of murine BMSCs induced by BMP-2
投稿时间：2014-03-20  修订日期：2014-03-29
DOI：10.11752/j.kqcl.2014.03.04
中文关键词:  微小核糖核酸  成骨分化  骨髓间充质干细胞  肿瘤坏死因子α  骨形态发生蛋白-2
英文关键词:miRNA  Osteogenic differentiation  BMSCs  TNF-α  BMP-2
基金项目:国家自然科学基金资助项目(81170988);上海市科学技术委员会基金资助项目(11ZR1420200)
作者单位E-mail
施琼玲 上海交通大学医学院附属第九人民医院口腔修复科, 上海市口腔医学重点实验室, 上海 200011  
吴婷婷 唾液腺疾病研究中心, 首都医科大学口腔医学院牙齿再生与功能重建重点实验室, 北京 100050  
李静 上海交通大学医学院附属第九人民医院口腔修复科, 上海市口腔医学重点实验室, 上海 200011  
汪艳 上海交通大学医学院附属第九人民医院口腔修复科, 上海市口腔医学重点实验室, 上海 200011  
黄慧 上海交通大学医学院附属第九人民医院口腔修复科, 上海市口腔医学重点实验室, 上海 200011 huanghui_68@126.com 
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中文摘要:
      目的探讨肿瘤坏死因子α(TNF-α)对骨形态发生蛋白-2(BMP-2)诱导下小鼠骨髓间充质干细胞(BMSCs)成骨分化的微小核糖核酸(micro ribonucleicacid,miRNA)表达谱的影响。方法给予小鼠BMSCs以下分组刺激:1阴性对照组(ctrl);2阳性对照组(pos):BMP-2(200ng/ml)刺激48h;3实验组(48h):BMP-2(200ng/ml)+TNF-α(10ng/ml)刺激48h;4实验组(72h):BMP-2(200ng/ml)+TNF-α(10ng/ml)刺激72h,48h和72h后分别提取RNA,应用miRNA芯片检测获得miRNA表达谱,筛选部分差异表达的miRNA进行荧光实时定量PCR(RT-PCR)验证。结果 miRNA表达谱分析结果表明,与阳性对照组相比,48h双因子刺激下检测到表达上调超过1.5倍的miRNA有44个,72h刺激下检测到22个miRNA,72h与48h相比,检测到24个表达上调超过1.5倍的miRNA,RT-PCR验证与芯片结果基本相符合。结论 miRNA参与调控TNF-α模拟炎症状态下BMP-2诱导BMSCs的成骨分化过程,且相关miRNA在TNF-α作用下表达上调,可能参与调控TNF-α的抑制成骨作用。由此可进一步解释炎性因子对成骨细胞分化的抑制机制,为发现新的药物靶点提供理论依据。
英文摘要:
      Objective In this study, we aimed to investigate the micro ribonucleicacid ( miRNA) expres-sion profile in the tumor necrosis factor (TNF)-α-inhibited osteogenic differentiation of bone marrow mesen-chymal stem cells(BMSCs) introduced by BMP-2. Methods The BMSCs was cultured with/without TNF-αand bone morphogenetic protein(BMP)-2 induction: ①negative control group(ctrl);② positive control(pos):stimulation with BMP-2(200ng/ml)for 48 h;③experimental group(48h):stimulation with BMP-2(200ng/ml) and TNF-α(10ng/ml)for 48 h; ④ experimental group(72h):stimulation with BMP-2(200ng/ml) and TNF-α(10ng/ml)for 72 h. Then the cells were harvested at the indicated times (48h, 72h). miRNA microar-ray technology was used to detect the expression profile of miRNA, and the expression of miRNA was verified by real time quantification-polymerase chain reaction (RT-PCR). Results Microarray analysis found that the expres-sion profile of miRNA changed dramatically. 44 miRNA were upregulated significantly more than 1.5-fold in 48h BMP-2/TNF-αgroup and 22 miRNA in 72h BMP-2/TNF-αgroup, compared with positive control group (P<0.01). Compared with 48h, 24 of up-regulated more than 1.5 times miRNA were detected in 72h BMP-2/TNF-αgroup, which were consistent with the results of miRNA microarray. Conclusion miRNA were involved in the BMP-2-induced osteogenic differentiation process with TNF-αto mimic a state of inflammation, and significantly upregulated miRNA may be involved in the suppression of osteogenesis by TNF-α. These findings can further explain the inhibitory mechanism of inflammatory cytokines on osteoblast differentiation and has important clinical significance.
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