核因子κB对人单核细胞诱导型一氧化氮合酶表达的调控作用

Nuclear factor κB regulates gene expression of inducible nitricoxide synthase in human monocyte

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?

AIM: To investigate whether gene expression of inducible nitric oxide synthase(iNOS)in human monocyte is modulated by nuclear factor κB(NF κB). METHODS: Human monocytes were cultured in medium with lipopolysaccharide(LPS)± pyrrolidine dithiocarbamate(PDTC)or in medium alone. One hour later, the nuclear protein of the monocytes was extracted for electrophoretic mobility shift assay (EMSA) and the monocyte coverslips were collected for immunofluorescence staining for NF κB p65 subunit. Ten hours later, RNA was extracted for RT PCR for iNOS. Twenty four hours later, whole cell extracts were used for Western blot and monocyte corverslips were collected for immunocytochemical staining. RESULTS: LPS increased the NF κB activation compared with that of the unstimulated cells. Ten hours later, the mRNA expression of iNOS increased and twenty four hours later the protein expression of iNOS increased markedly. The inhibition of NF κB activation by PDTC resulted in a significant decrease of the expressions of iNOS mRNA and protein hours later. The positive rate of nuclear staining of p65 and the DNA binding activity of NF κB significantly were correlated with the mRNA and protein expression of iNOS. CONCLUSION: NF κB regulates the gene expression of iNOS in human monocytes.