【摘要】 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。更多还原

【Abstract】 Objective To study the synergistic responses of nitric oxide synthase (NOS) induction in rat hepatocytes to LPS and various cytokines in vitro and the kinetic characteristics of iNOS. Methods The hepatocytes were isolated by in-situ pre-perfusion and collagenase circulatory perfusion of rat livers. The effects of LPS associated with IFN-r, TNF a and IL-1 βor IL-6 on NOS activity, cGMP, and NO2-+NO3- were observed in hepatocytes, respectively. Also the kinetic characteristics of this enzyme and dose response of corticosteroids on the induction of iNOS were analyzed. Results The maximum induction of NOS activity was observed in hepatocytes treated by LPS in combination with IFN-r, TNF αand IL-1 Вor IL-6. The kinetic analysis of this iNOS demonstrated specific constants of Km=10.8 u mol/L,Vmax=263.2 pmol. min-1·mg-1 protein(for L-Arg), and Ki of 0.56, 0.94 u mol/L for competitive inhibitor, L-NMMA and NNA, respectively. The time course of induction showed that iNOS activity peaked at 9 h; however, significant increase in release of NO2-+ NO3 and cGMP sustained for at least 18 h. Dexamethasone and hydrocortisone dramatically inhibited the NOS induction in hepatocytes in vitro with IC50 of 3.5 x 10-8 mol/L and 2.6 x 10-6 mol/L, respectively. Conclusions The expression of inductive NOS in hepatocytes requires specific synergetic action of cytokines, and the inducible characteristics may play an important pathogenesis in endotoxemia and septic shock.更多还原