旨为探明microRNA-199a(miR-199a)对脂肪细胞凋亡的影响及核转录因子 κB(nuclear factorκB,NF-κB)在其中的作用.培养3T3-L1脂肪细胞,使用肿瘤坏死因子 α(tumor necrosis factorα,TNFα)诱导细胞凋亡.在此基础上分别使用NF-κB阻断剂PDTC和miR-199a mimic处理细胞,判断过表达miR-199a对TNFα 诱导的脂肪细胞凋亡的影响及NF-κB在其中的作用.使用流式细胞仪分析细胞凋亡率,试剂盒检测Caspase3/7酶活,双荧光素酶报告系统检测NF-κB活性,qRT-PCR检测miR-199a的表达水平,Western blotting检测NF-κB相关蛋白变化.结果显示,TNFα 可以诱导分化的脂肪细胞凋亡并同时激活NF-κB,激活的NF-κB在TNFα 诱导的脂肪细胞凋亡中发挥负调控作用.在脂肪细胞过表达miR-199a明显抑制NF-κB的激活进而显著促进TNFα诱导的凋亡.miR-199a通过调控NF-κB活性参与TNFα 诱导的脂肪细胞凋亡.%The aim of this study is to explore the effects of MicroRNA-199a(miR-199a)on adipocytes apoptosis and the role of nuclear factor κB(NF-κB). Tumor necrosis factor α(TNFα)was used to induce the apoptosis of cultured 3T3-L1 adipocytes. Based on this work, adding PDTC of NF-κB blocker and miR-199a mimic,the effect of overexpression of miR-199a on TNFα-induced apoptosis and the role of NF-κB in it were determined. The flow cytometry was used to analyze the cell apoptosis,kit Caspase3/7 to detect the enzymatic activities,dual luciferase reporter assay to detect the activity of NF-κB,qRT-PCR to detect the expression of miR-199a,and Western blotting to detect the changes of relevant proteins. As results showed,TNFα induced the apoptosis of differentiated adipocytes,concurrently activated NF-κB. The activated NF-κB played a negative regulator role in the apoptosis of adipocytes mediated by TNFα. Over-expressed miR-199a significantly suppressed the activation of NF-κB,and then remarkably promoted the apoptosis induced by TNFα. Conclusively,miR-199a involves in the TNFα-induced apoptosis of adipocytes through regulating NF-κB activation.
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