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目的 :探讨人单核细胞诱导型一氧化氮合酶 (iNOS)的表达是否受核因子κB(NF κB)调控 .方法 :人单核细胞分别给予脂多糖 (LPS)、LPS加二硫代氨基甲酸吡咯烷 (PDTC)、空白对照刺激培养 .提取核蛋白进行NF κB的电泳移动迁移率改变试验 (EMSA) ;提取RNA进行iNOS的RT PCR ;提取总蛋白进行iNOS的Westernblotting ;并留细胞爬片进行p6 5亚基的免疫荧光检测和iNOS的免疫酶学检测 .结果 :LPS刺激 1h后 ,单核细胞p6 5核表达阳性率及核蛋白NF κB的DNA结合活性均较空白刺激组明显增加 (P <0 .0 5 ) ,而LPS +PDTC组p6 5核表达阳性率及NF κB的DNA结合活性均较LPS组明显降低 (P <0 .0 5 ) ;LPS刺激 1 0h后 ,单核细胞iNOSmRNA的表达较空白刺激组明显增加 (P <0 .0 5 ) ,而LPS +PDTC组iN OSmRNA表达较LPS组明显降低 (P <0 .0 5 ) ;LPS刺激 2 4h后 ,单核细胞iNOS蛋白表达量 (不论是Westernblot,还是免疫组化结果 )均较未刺激组明显增加 (P <0 .0 5 ) ,而LPS +PDTC组均较LPS组明显降低 (P <0 .0 5 ) ;NF κB核表达阳性率与iNOSmRNA、蛋白表达量成正相关 ;NF κB的DNA结合活性亦与iN OSmRNA和蛋白表达量成正相关 .结论 :NF κB的激动剂能促进人单核细胞iNOS的表达 ,且这种促进作用是首先出现在mRNA水平 ,而NF κB的抑制?
Objective: To investigate whether the expression of inducible nitric oxide synthase (iNOS) is regulated by nuclear factor κB (NF κB) in human monocytes.Methods Human monocytes were treated with lipopolysaccharide (LPS), LPS plus dithioamino (PDTC) and blank control to stimulate the culture.Nuclear proteins were extracted for electrophoretic mobility shift assay (EMSA) of NF-κB; RNA was extracted for RT-PCR of iNOS; total protein was extracted for iNOS Western blotting; Immunofluorescence assay and iNOS immunodetection assay were used to detect the expression of p65 subunit.Results: The positive rate of p65 nuclear expression in monocytes and the DNA binding activity of NFκB were significantly increased P <0.05). However, the positive rate of p65 nuclear expression and the DNA binding activity of NF-κB in LPS + PDTC group were significantly lower than those in LPS group (P <0.05). After LPS stimulation for 10h, monocytes Compared with LPS group, the expression of iNOS mRNA was significantly increased (P <0.05), while the expression of iNOS mRNA in LPS + PDTC group was significantly lower than that in LPS group (P <0.05). After LPS stimulation for 24 hours, the expression of iNOS Protein expression (whether Westernblot or immunohistochemistry results) were significantly increased compared with the unstimulated group (P <0.05), while LPS + PDTC group was significantly lower than LPS group (P <0.05). The positive rate of NF-κB expression was positively correlated with the expression of iNOS mRNA and protein. The NF-κB DNA binding activity Also positively correlated with iN OS mRNA and protein expression.Conclusion: NF κB agonist can promote iNOS expression in human monocytes, and this promoting effect is first appeared at the mRNA level, while NF κB inhibition?