瘦素对IL-1β诱导大鼠骨性关节炎软骨细胞 MMP-13mRNA表达的影响-东南大学学报医学版

瘦素对IL-1β诱导大鼠骨性关节炎软骨细胞 MMP-13mRNA表达的影响

单位：1. 中国医科大学附属盛京医院骨外科, 辽宁沈阳 110004;
2. 辽宁医药职业学院卫生检验教研室, 辽宁沈阳 110010;
3. 中国医科大学公共卫生学院营养与食品卫生学教研室, 辽宁沈阳 110122

分类号：R-332;R684.3

出版年·卷·期（页码）：2015·34·第六期（870-875）

摘要：

目的:探讨瘦素对IL-1β诱导大鼠骨性关节炎软骨细胞基质金属蛋白酶-13 (MMP-13)mRNA表达的影响及其机制.方法:体外培养大鼠膝关节软骨细胞,取第3代细胞采用免疫细胞化学方法及甲苯胺蓝染色法鉴定 Ⅱ 型胶原及蛋白多糖表达;MTT法检测不同质量浓度瘦素环境下正常及IL-1β诱导软骨细胞的增殖活力;实时定量PCR法检测不同质量浓度瘦素处理后IL-1β诱导软骨细胞MMP-13 mRNA的表达, ELISA法检测培养基上清肿瘤坏死因子α(TNFα)的水平;通过小RNA干扰(siRNA)抑制瘦素长型受体(OB-Rb)表达,观察瘦素对MMP-13 mRNA表达的影响.结果:所有细胞均能分泌 Ⅱ 型胶原及蛋白多糖.MTT结果显示,低质量浓度瘦素对细胞增殖无明显影响,瘦素质量浓度大于等于10 ng·ml^-1后可显著促进细胞增殖;而10 ng·ml^-1的 IL-1β可显著抑制细胞增殖,同时给予100 ng·ml^-1及1 μg·ml^-1瘦素则可进一步抑制细胞增殖,差异均有统计学意义(P<0.05).实时定量PCR结果显示,IL-1β可增加软骨细胞MMP-13 mRNA表达及TNFα分泌,而加入瘦素(100 ng·ml^-1)后可进一步刺激MMP-13 mRNA的表达及TNFα的分泌,差异具有统计学意义(P<0.05).转染OB-Rb siRNA的软骨细胞OB-Rb mRNA的表达显著下降(P<0.05),MMP-13 mRNA的表达则不受影响;IL-1β处理对转染组OB-Rb的 mRNA表达无明显影响,但可显著诱导MMP-13 mRNA的表达增加(P<0.05),进一步加入瘦素后,OB-Rb mRNA的表达有所增加,但表达量远远低于siRNA未处理组,差异具有统计学意义(P<0.05),MMP-13 mRNA的表达则未见增加.结论:一定浓度的瘦素可通过与OB-Rb结合促进IL-1β诱导大鼠骨性关节炎软骨细胞MMP-13 mRNA表达,具有降解软骨作用.

Objective: To determine the effect and mechanism of leptin on the expression of matrix metalloproteinase 13 (MMP-13) mRNA in IL-1β-induced rat osteoarthritis articular chondrocytes. Methods: Rat articular chondrocytes in generation 3 were identified by immunocytochemistry and toluidine blue staining. Chondrocytes were incubated with leptin in the absence/presence of IL-1β. Cell viability was evaluated using the MTT assay. The expression of MMP-13 mRNA in chondrocytes and TNFα levels in culture supernatants were measured by real-time RT-PCR and ELISA, respectively. In addition, long-form leptin receptor (OB-Rb) siRNA was used to block OB-Rb expression and the effect of leptin on MMP-13 expression was detected. Results: All cultured chondrocytes expressed type 2 collagen and proteoglycan. MTT assay showed that leptin at low concentration had no effect on cell viability, but leptin at high concentration (≥10 ng·ml^-1) could significantly promote cell proliferation. Viability of cells exposed to IL-1β (10 ng·ml^-1) was significantly impaired, and it was further inhibited in the presence of leptin at 100 ng·ml^-1 and 1 μg·ml^-1 (P<0.05). The expression of MMP-13 mRNA and levels of TNF-α were significantly upregulated in the presence of IL-1β, and leptin (100 ng·ml^-1) cotreatment could further stimulate MMP-13 mRNA expression and TNF-α level (P<0.05). The expression of OB-Rb mRNA was significantly inhibited, but MMP-13 expression was not affected in those chondrocytes transfected by OB-Rb siRNA. IL-1β treatment could significantly increase MMP-13 expression, but not OB-Rb mRNA in the OB-Rb knock-down cells (P<0.05). The expression of OB-Rb mRNA in IL-1β-treated OB-Rb siRNA cells was dramatically lower than that of untransfected cells (P<0.05) after further addition of leptin, while MMP-13 expression was not affected. Conclusion: Leptin could stimulate MMP-13 expression via OB-Rb in IL-1β-induced rat osteoarthritis articular chondrocytes, which demonstrated that leptin might play an important role in cartilage degradation.

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