瘦素对IL-1β诱导大鼠骨性关节炎软骨细胞MMP-13 mRNA表达的影响  

Effect of leptin on the expression of MMP-13 mRNA in IL-1β-induced rat osteoarthritis articular chondrocytes

作　　者：顾海伦[1] 刘亚莉[2] 王维[1] 丁立峰[1] 刘莉[3] 

机构地区：[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

出　　处：《东南大学学报（医学版）》2015年第6期870-875,共6页Journal of Southeast University(Medical Science Edition)

基　　金：国家自然科学基金资助项目(81372971);辽宁省自然科学基金资助项目(201202267)

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1μg·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 metalloproteinase13（ 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 at100 ng·ml- 1and 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,whic

关 键 词：瘦素 骨性关节炎 软骨细胞 基质金属蛋白酶-13 瘦素长型受体 大鼠 

分 类 号：R-332[医药卫生] R684.3