TNF-a对BMP-2诱导下小鼠BMSCs成骨分化miRNA表达的影响  

Effect of TNF-α on the miRNA expression profile in osteogenic differentiation of murine BMSCs induced by BMP-2

作　　者：施琼玲[1] 吴婷婷[2] 李静[1] 汪艳[1] 黄慧[1] 

机构地区：[1]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 [2]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

出　　处：《口腔材料器械杂志》2014年第3期130-136,共7页Chinese Journal of Dental Materials and Devices

基　　金：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)

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。Objective In this study, we aimed to investigate the micro ribonucleicacid （miRNA） expression profile in the tumor necrosis factor （TNF）- α -inhibited osteogenic differentiation of bone marrow mesenchymal stem cells（BMSCs） introduced by BMP-2. Methods The BMSCs was cultured with/without TNF- α and bone morphogenetic protein（BMP）-2 induction： （1） negative control group （ctrl）： （2） positive control （pos）- stimulation with BMP-2 （200ng/ml） for 48 h; （3） experimental 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 microarray 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 expression profile of miRNA changed dramatically. 44 miRNA were upregulated significantly more than 1.5-fold in 48h BMP-2/TNF-α group and 22 miRNA in 72h BMP-2/TNF-α group, 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-α group, which were consistent with the results of miRNA microarray. Conclusion miRNA were involved in the BMP-2- induced osteogenic differentiation process with TNF-α to 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.

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 

分 类 号：R782.2[医药卫生—口腔医学]