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目的 探讨脂多糖 (LPS)诱导单核 巨噬细胞诱导型一氧化氮合酶 (iNOS)基因表达的分子机制。方法 用LPS刺激诱导RAW2 64 .7细胞 ,观察其对蛋白激酶C(PKC)的激活 ,用Griess还原法测定PKC抑制剂对LPS诱导的一氧化氮 (NO)生成作用 ,并用逆转录PCR(RT PCR)研究其对iNOS基因表达的影响 ;构建iNOS荧光素酶报告基因载体 ,用基因转染及报告基因检测等方法研究LPS刺激RAW2 64 .7细胞对iNOS启动子活性的诱导作用及PKC对其影响。结果 LPS刺激RAW2 64 .7细胞引起PKC磷酸化激活和膜移位 (P <0 .0 1 ) ;LPS刺激RAW2 64 .7细胞 1 2h后即可明显诱导NO生成 (30 .1μmol L± 5 .4μmol L ,P <0 .0 1 )和iNOS基因表达 ;PKC抑制剂H 7可抑制NO的生成 (P <0 .0 1 )和iNOS基因表达 ;LPS刺激诱导iNOS启动子的转录活性 [(2 5 .3± 4 .6)相对荧光素酶活性单位 ,P <0 0 1 ] ,用H 7和钙离子通道阻滞剂异搏定均可抑制其转录活性 (P <0 .0 1 )。结论 LPS刺激RAW2 64 .7细胞 ,通过引起细胞内钙增加和PKC激活诱导iNOS基因表达 ,使NO生成增加 ,这是内毒素休克时NO增加的一个重要的信号机制
Objective To investigate the molecular mechanism of inducible nitric oxide synthase (iNOS) gene expression in monocytes induced by lipopolysaccharide (LPS). Methods RAW264.7 cells were induced by LPS and the activation of protein kinase C (PKC) was observed. The effect of PKC inhibitor on LPS-induced nitric oxide (NO) production was determined by Griess reduction assay. PCR) to study its effect on iNOS gene expression; iNOS luciferase reporter gene vector was constructed, using gene transfection and reporter gene detection methods to study LPS stimulation of RAW264.7 cells on iNOS promoter activity induction and PKC influences. Results LPS stimulated PKC phosphorylation and membrane translocation in RAW264.7 cells (P <0.01). LPS stimulated RAW264.7 cells to produce NO (30.1μmol L ± 5. 4μmol L, P <0.01) and iNOS gene expression; PKC inhibitor H 7 inhibited NO production (P <0.01) and iNOS gene expression; LPS stimulated the transcriptional activity of iNOS promoter [(2 5 .3 ± 4 .6) relative luciferase activity units (P <0.01). Both H 7 and calcium channel blockers were able to inhibit the transcriptional activity of verapamil (P <0.01). Conclusion LPS stimulates RAW264.7 cells to increase the production of NO by inducing the increase of intracellular calcium and activation of PKC, which is an important signal mechanism of NO increase during endotoxic shock