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目的对内毒素和几种细胞因子诱导肝细胞一氧化氮合酶的协同效应及酶动力学参数进行研究。方法原位预灌流和胶原酶循环灌流大鼠肝脏、分离肝实质细胞，观察内毒素、 ＩＦＮ－ γ、 ＩＦＮ－ α、 ＴＮＦ α、 ＩＬ－１β、ＩＬ－６及不同组合对肝细胞一氧化氮合酶活性、ｃＧＭＰ及ＮＯ２－＋ＮＯ３－的影响，分析酶动力学特征及皮质甾与酶诱导的量效关系。结果 内毒素＋ＩＦＮ－γ＋ＴＮＦα＋ＩＬ－１β（ＩＬ－６）组台诱导酶表达效应最显著；酶参数分析显示：Ｋｍ、 Ｖｍａｘ分别为 １０．８ μ ｍｏｌ／Ｌ和 ２６３．２ ｐｍｏｌ·ｍｉｎ－１· ｍｇ－１蛋白质，竞争性抑制剂 Ｌ－ＮＭＭＡ、 Ｌ－ＮＮＡ作用的 Ｋｉ分别为 ０．５６ ｕ ｍｏｌ／Ｌ及 ０．９４ ｕ ｍｏｌ／Ｌ；诱导时间进程显示： ｉＮＯＳ活性表达在 ９ｈ达到峰值，但ｃＧＭＰ及 ＮＯ２－＋ＮＯ３－的释放持续增加可维持至 １８ ｈ；地塞米松和氢化可的松抑制肝细胞酶诱导的 ＩＣ５０分别为 ３．５ ×１０－８ｍｏｌ／Ｌ和２．６ × １０－６ｍｏｌ／Ｌ。结论肝细胞诱导性一氧化氮合酶的表达依赖特异多细胞因子协同作用，这种可诱导性特征可能在内毒素血症和败血症休克发病机制中具有重要意义。
Objective To study the synergistic effect of endotoxin and several cytokines on the induction of nitric oxide synthase (NOS) in hepatocytes and the kinetic parameters of the enzyme. Methods The hepatic cells were perfused by collagenase in situ and perfused in situ. Hepatic parenchymal cells were isolated and the effects of endotoxin, IFN-γ, IFN-α, TNFα, IL-1β, IL- Synthase activity, cGMP and NO2- + NO3-, the enzyme kinetic characteristics and the dose-response relationship between corticosteroid and enzyme were analyzed. Results The enzyme-induced expression of endotoxin + IFN-γ + TNFα + IL-1β (IL-6) was the most significant. Enzyme parameters analysis showed that Km and Vmax were 10.8 μ mol / L and 263.2 pmol · min-1 · mg -1 protein and competitive inhibitors L-NMMA, L-NNA Ki were 0.56 u mol / L and 0.94 u mol / L; induction time course showed that: iNOS activity peaked at 9h, but The release of cGMP and NO2- + NO3- continued to increase up to 18 h. The IC50 of dexamethasone and hydrocortisone in inhibiting hepatic cell enzyme induction were 3.5 × 10 -8 mol / L and 2.6 × 10 -6 mol / L. Conclusion The expression of inducible nitric oxide synthase in hepatocytes relies on the synergistic effect of specific cytokines. This inducible feature may play an important role in the pathogenesis of endotoxemia and septic shock.