【摘要】 目的探讨CCK-8抑制LPS诱导ECV-304 iNOS表达的分子机制。方法培养ECV-304,用溶剂、CCK-8、LPS及CCK受体拮抗剂丙谷胺分别或联合孵育细胞,用W estern b lot检测iNOS蛋白表达,用免疫细胞化学方法检测NF-κB P65蛋白表达与核移位,用RT-PCR检测CCK-AR和CCK-BR mRNA表达。结果①溶剂对照组ECV-304 iNOS蛋白呈低表达;LPS诱导iNOS表达明显上调,CCK-8剂量依赖性抑制LPS的作用,单独CCK-8对其无影响。②ECV-304存在CCK-AR和CCK-BR mRNA表达,其中CCK-BR mRNA表达稍强。LPS可诱导CCK-AR和CCK-BR mRNA表达明显增强。③溶剂对照组NF-κB P65蛋白主要分布于细胞质;LPS孵育1 h后细胞核中NF-κB P65表达明显增加,CCK-8剂量依赖性抑制LPS的作用;单独CCK-8对其无影响。④丙谷胺可翻转CCK-8的抑制作用。结论ECV-304存在CCK-AR和CCK-BR mRNA表达;CCK受体和NF-κB参与介导CCK-8抑制LPS诱导ECV-304 iNOS蛋白表达上调。更多还原

【Abstract】 Objective To elucidate the molecular mechanism of cholecystokinin octapeptide（CCK-8） inhibiting of iNOS expression in ECV-304 induced by LPS.Methods ECV-304 cells,were stimulated with LPS in the presence or absence of CCK-8(10^-9-10^-7mol/L) or/and CCK receptor antagonist proglumide.The iNOS protein level in the cytoplasm at 16 h after stimulation was detected with Western blotting.The expressions of CCK-AR and CCK-BR mRNA were assayed with RT-PCR.The level of NF-κB P65 protein was detected in cytoplasm and nuclus at 1 h after stimulation with immunocytochemical technique.Results LPS led to the up-regulation of iNOS protein expression in ECV-304 that was obviously inhibited by CCK-8 in a dose-dependent manner.CCK-8 alone did not affect iNOS protein expression.CCK-AR mRNA expression was higher than CCK-BR mRNA expression in ECV-304.The expression of CCK-AR and CCK-BR mRNA could be up-regulated obviously by LPS.LPS increased NF-κB P65 protein expression in the nuclus of ECV-304.CCK-8 obviously inhibited LPS-induced changes of NF-κB P65 protein in a dose-dependent manner.The inhibitive effects of CCK-8 on iNOS protein expression and NF-κB P65 were attenuated by proglumide.Conclusion There were CCK-AR and CCK-BR mRNA expressions in ECV-304.CCK-8 inhibited LPS-induced up-regulation of iNOS protein expression by acting CCK receptors and NF-κB in ECV-304.更多还原