汪立梅，张家明，吕正涛，向威，王胜洁，吴颖星，张津铭，郭风劲，许涛.机械应力调控软骨细胞炎症反应中长链非编码RNA的机制研究[J].中华物理医学与康复杂志,2017,39(2):86-91
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| 机械应力调控软骨细胞炎症反应中长链非编码RNA的机制研究 |
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| DOI： |
| 中文关键词: 软骨细胞 机械应力 自噬 MEG3 |
| 英文关键词: Chondrocytes Mechanical strain Autophagy LncRNA-MEG3 |
| 基金项目:国家自然科学基金（81371915, 81572094） |
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| 中文摘要: |
| 目的探讨机械应力调控软骨细胞炎症反应的作用机制。 方法体外分离培养新生SD大鼠软骨细胞，经白介素-1β（IL-1β）干预构建软骨细胞炎症模型，设置对照组（IL-1β）、2000μstrain组（2000μstrain应力+IL-1β）、5000μstrain组（5000μstrain应力+IL-1β），IL-1β浓度为5ng/ml，以1Hz应力干预2h。采用Real-time PCR和Western Blot检测Ⅱ型胶原（Col-2）、基质金属蛋白酶13（MMP13）、自噬标记蛋白LC3、Beclin-1及m-TOR表达，同时检测MEG3表达，采用免疫荧光染色检测自噬小体形成；si-RNA沉默MEG3后，采用Real-time PCR检测LC3及Beclin-1表达。 结果2000μstrain组软骨细胞Col-2、LC3、Beclin-1基因及Beclin-1蛋白表达量均较对照组及5000μstrain组明显上调（P<0.05）；2000μstrain组软骨细胞MEG3基因表达量较对照组及5000μstrain组明显下调（P<0.05）；5000μstrain组m-TOR蛋白表达较对照组及2000μstrain组明显增加（P<0.05）；应力组自噬标记蛋白LC3Ⅱ/Ⅰ相对表达量均较对照组明显减少（P<0.05）；免疫荧光染色显示2000μstrain组自噬小体形成较对照组及5000μstrain组明显增加，5000μstrain组自噬小体形成较对照组及2000μstrain组明显减少（P<0.05）；si-RNA沉默MEG3后，发现LC3及Beclin-1表达量均明显增加（P<0.05）。 结论机械应力可影响软骨细胞LncRNA-MEG3表达，从而调控炎症环境中软骨细胞自噬水平。 |
| 英文摘要: |
| Objective To explore the mechanism by which mechanical strain regulates the inflammatory responses of chondrocytes. MethodsChondrocytes were harvested from newborn Sprague-Dawley rats and cultured in vitro. The chondrocytes were subjected to 2000μstrain or 5000μstrain mechanical strain at a frequency of 1 Hz for 2 h. Real-time PCR and western blotting were used to detect the expression of collagen type Ⅱ (Col-2), matrix metalloproteinase (MMP13), and autophagy marker proteins LC3 and Beclin-1. The expression of LncRNA-MEG3 was detected simultaneously. Immunofluorescence was used to detect the expression of LC3 and Beclin-1 after real-time PCR when LncRNA-MEG3 had been silenced by si-RNA. ResultsThe expression of the Col-2 and LC3 genes was significantly up-regulated in IL-1β-treated chondrocytes subjected to 2000μstrain mechanical strain. The level of the autophagy marker protein LC3Ⅱ/Ⅰ in the strained group was significantly higher than that in the control group. The expression of Beclin-1 and m-TOR in the strain-stressed group was significantly higher than in the control group. Immunofluorescence staining showed that the expression of LC3 in the 2000μstrain group was significantly higher than that in the control group, while in the 5000μstrain group it was significantly lower. The expression of LC3 was significantly increased after silencing LncRNA-MEG3 using si-RNA. ConclusionMechanical strain regulates the autophagy of chondrocytes in an inflammatory environment by regulating the expression of LncRNA-MEG3. |
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