摘要
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核转位及结合活性的变化。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无明显抑制作用。变异型NIK及IKK能显著降低报告基因荧光素酶的活性，mm-LDL激活的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。
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 (E- MSA)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 α(IKKα)and inhibitor of NF-kB kinase β(IKKβ) 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-IKKβ inhibited lucifer-ase activity induced by mm-LDL． mm-LDL could also enhance luciferase activity controled by upstr-eam 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-IKKβ pathway and promote PDGFb mRNA expression in endothelial cells．
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2001年第4期312-316,共5页
Acta Academiae Medicinae Sinicae
基金
国家自然科学基金重点项目(39730220)资助
