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目的 :探讨TNF α启动子中 3个κB位点在调节基因转录中的作用及其与核蛋白作用的亲和力关系。方法 :用含不同调节区域的重组报告基因进行HL 6 0细胞的基因转染 ,检测LPS刺激前后及κB3 反义寡核苷酸封闭后的报告基因表达水平 ;提取LPS刺激 6h的HL 6 0的细胞核蛋白 ,用凝胶迁移率改变实验及竞争结合实验比较NF κB与TNF α的 3个NF κB位点的亲和力。结果 :尽管TNF α基因中 3个κB位点都参与该基因的诱导和非诱导性转录调节 ,但κB3 位点的作用更为重要 ;3个κB位点能同LPS刺激和非刺激的HL 6 0细胞核蛋白发生特异性结合 ,但LPS刺激的蛋白 DNA复合物电泳条带明显增粗 ,尤其κB3 位点出现 2条明显的特异性条带 ;3个κB位点与LPS诱导后HL 6 0核蛋白的亲和力强弱顺序依次为 :κB2 >κB1 >κB3。结论 :TNF α基因的 3个κB位点都能与NF κB结合 ,从而参与调节HL 6 0细胞TNF α组成性表达和LPS诱导性表达。尽管κB3 位点在对LPS刺激的反应性中发挥的作用最大 ,但这种效应与其同核蛋白亲和力的大小无关。
OBJECTIVE: To investigate the role of three kappa B sites in TNF alpha promoter in regulating gene transcription and its affinity with nucleoprotein. Methods: The gene of HL-60 cells was transfected with recombinant reporter genes containing different regulatory regions to detect the expression of reporter gene before and after LPS stimulation and the blocking of κB3 antisense oligonucleotide. Nucleoprotein was compared for the affinity of NFκB with the three NFκB sites of TNFα using a gel shift assay and a competition binding assay. RESULTS: Although all three kappa B sites in TNFα gene were involved in the induction and non-inducible transcriptional regulation of the gene, the role of κB3 site was more important. The three kappa B sites could interact with LPS-stimulated and non-stimulated HL6 0 nucleus protein, but LPS-stimulated protein DNA complex electrophoresis bands were significantly thicker, especially in the κB3 site showed two distinct specific bands; 3 kappaB sites and LPS-induced HL 60 nuclei The order of affinity of protein is: κB2> κB1> κB3. CONCLUSION: All three TNFα genes can bind to NF κB, which may be involved in the regulation of TNFα constitutive expression and LPS induction in HL-60 cells. Although the κB3 site plays the largest role in reactivity to LPS stimulation, this effect is not related to its affinity for nuclear proteins.