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| TNF-a对BMP-2诱导下小鼠BMSCs成骨分化miRNA表达的影响 |
| miRNA expression profile in the TNF-a-inhibited osteogenic differentiation of murine BMSCs introduced by BMP-2 |
| 投稿时间：2014-03-20 修订日期：2014-03-21 |
| DOI： |
| 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-a 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)。 |
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| 中文摘要: |
| 目的：本研究旨在探讨肿瘤坏死因子α(tumor necrosis factor-a, TNF-a)对骨形态发生蛋白-2(bone morphogenetic protein-2, BMP-2)诱导下小鼠骨髓间充质干细胞(bone marrow stromal cell, 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48小时；③实验组(48h)：BMP-2(200ng/ml) TNF-a(10ng/ml)刺激48小时；④实验组(72h)：BMP-2(200ng/ml) TNF-a(10ng/ml)刺激72小时,48h和72h后分别提取RNA,应用miRNA芯片检测分析获得miRNA表达谱,筛选部分差异表达的miRNA进行荧光实时定量PCR(real-time quantitative polymerase chain reaction, 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-a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-a作用下表达上调，可能参与调控TNF-a的抑制成骨作用。这一发现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，具有重要的临床意义。 |
| 英文摘要: |
| Objective: In this study, we aimed to investigate the miRNA expression profile in the TNF-a-inhibited osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs) introduced by BMP-2. Methods: The BMSCs was cultured with/without TNF-a and BMP-2 induction ：①negative control group（ctrl）；② positive control（pos）：introduced by BMP-2（200ng/ml）for 48 hours；③experimental group（48h）：stimulation with BMP-2（200ng/ml） and TNF-a（10ng/ml）for 48 hours；④experimental group（72h）：stimulation with BMP-2（200ng/ml） and TNF-a（10ng/ml）for 72 hours，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-a group and 22 miRNA in 72h BMP-2-TNF-a group compared with positive control group (P＜0.01), Furthermore, the expressions of miR- 210-3p、 miR-33-5p and so on were verified by RT-PCR and found they both increased progressively over time after stimulation, and were content with the results of miRNA microarray. Conclusion: miRNA were involved in the the BMP-2-induced osteogenic differentiation process under the inhibitation of TNF-a, and significantly upregulated miRNA may be involved in the suppression of osteogenesis by TNF-a.and these findings can further explain the inhibitory mechanism of inflammatory cytokines on osteoblast differentiation. That have important clinical significance. |
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