mm—LDL对内皮细胞转录因子NF—kB活性的影响

目的 研究轻微修饰低密度脂蛋白（mm-LDL）激活内皮细胞转录因子NF－kB的信号传导途径以及NF－kB对血小板源性生长因子B链（PDGFb）表达的调控作用。方法 以FeSO4修饰法制备mm-LDL,以正常LDL（n-LDL）作对照,作用于培养内皮细胞后提取核蛋白,用凝胶阻滞实验检测转录因子NF－kB停车场 及结合活性的变化。观察自由基清除剂probucol及PDTC对mm-LDL激活内皮细胞转录因子NF－kB的影响。通过检测报告基因探讨核因子诱导激酶（NIK）和核因子－kB抑制亚基激酶（IKK）在激活内皮细胞转录因子NF－kB的信号传导途径中的作用以及mm-LDL激活的NF－kB对PDGFb基因启动子的作用。结果 mm-LDL能激活培养内皮细胞转录因子NF－kB,自由基清除剂probucol和PDTC对mm-LDL激活内皮细胞转录因子NF－kB还能增加带有PDGFb基因上游调控序列（－189／＋43)的报告基因荧光素酶的活性。Slot blot结果显示变异型NIK能显著减少受mm-LDL刺激的内皮细胞PDGFb mRNA含量。结论 mm-LDL可通过NIK－IKK途径激活内皮细胞转录因子NF－kB并促进PDGFb基因表达。

Effect of mm-LDL on NF-kB activation in endothelial cell

OBJECTIVE: To investigate the signal transduction pathway of NF-kB activated by minimally modified low density lipoprotein (mm-LDL) in endothelial cells and the effect of NF-kB on platelet derived growth factor b (PDGFb) mRNA expression. METHODS: mm-LDL was prepared through iron oxidation by dialyzing the native LDL against FeSO4 in PBS. Endothelial cells were incubated in a medium containing mm-LDL, TNF, and IL-1 respectively and electrophoretic mobility shift assay (EMSA) was displayed to check on the activation of NF-kB. Luciferase reporter gene was analysed to investigate the effect of nuclear factor inducing kinase (NIK), inhibitor of NF-kB kinase alpha (IKK alpha) and inhibitor of NF-kB kinase beta (IKK beta) on NF-kB activation. In addition, endothelial cells were transfected using PDGFb promoter-luciferase for reporter gene analysis or transfected with mut-NIK for slot blot analysis to study the effect of NF-kB on PDGFb mRNA expression. RESULTS: mm-LDL was able to activate NF-kB in endothelial cells. mut-NIK and mut-IKK beta inhibited luciferase activity induced by mm-LDL. mm-LDL could also enhance luciferase activity controlled by upstream sequence of PDGFb promoter which contains element interacting with NF-kB. Result of slot blot showed inhibition of PDGFb mRNA expression by mut-NIK in the endothelial cells stimulated by mm-LDL. CONCLUSION: mm-LDL may activate NF-kB through NIK-IKK beta pathway and promote PDGFb mRNA expression in endothelial cells.