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目的 研究肝衰竭病理过程中血氨的代谢特点及其血氨通过氨基甲酰磷酸合酶Ⅱ(CPS-Ⅱ)途径的基本机制,探索血氨升高对肝细胞损伤的基本规律,为高血氨在肝衰竭发生中的分子生物学机制提供理论基础. 方法 体外构建Changliver细胞高血氨模型,设定不同梯度浓度的氯化铵(NH4Cl)干预Changliver细胞,显微镜观察肝细胞形态变化,流式细胞仪检测细胞凋亡率,RT-PCR检测CPS-Ⅱ基因mRNA相对表达量,Western blot检测CPS-Ⅱ蛋白表达.计量资料采用t检验,计数资料采用x2检验进行统计学分析.结果 与对照组相比,随着氨浓度的升高,凋亡的Changliver细胞明显增多.流式细胞仪检测对照组Changliver细胞的凋亡率为4.3％,与1 mmol/L组的6.1％比较,x 2=2.931,P=0.0869,差异无统计学意义;与5 mmol/L组的18.3％和10mmol/L的24.7％比较,x2值分别为96.382、166.2,P值均＜0.000 1,差异均有统计学意义.RT-PCR检测1 mmol/L组、5 mmol/L组和10 mmol/L组CPS-ⅡmRNA的相对表达量分别为1.641±0.123、2.285±0.167、3.347±0.124,与对照组的1.040±0.045比较,F=191.881,P＜ 0.000 1,差异有统计学意义.Western blot检测1 mmol/L组、5 mmol/L组和10 mmol/L组CPS-Ⅱ蛋白相对表达量分别为0.143±0.025、0.161±0.036和0.223±0.042,均明显高于对照组的0.099±0.0130,t值分别为3.492、3.622、6.307,P值均＜0.05,差异均有统计学意义.结论 随着Changliver细胞外氨浓度升高,在损伤肝细胞中CPS-Ⅱ mRNA和蛋白的表达水平明显增加,表明在高氨状态下除鸟氨酸循环外,CPS-Ⅱ在氨代谢中可能发挥着重要作用.“,”Objective To study the CPS-Ⅱ mechanism underlying the pathological process of elevated blood ammonia leading to liver injury.Methods An in vitro hyperammonemia hepatocyte cell model was constructed by exposure to various concentrations of NH4Cl.The subsequent changes to cellular morphology were observed by microscopy.to cell apoptosis were determined by flow cytometry,and to mRNA and protein expression of CPS-Ⅱ were examined by real-time PCR and western blotting,respectively.Results Exposure to NH4Cl led to dose-dependent morphological damage,apoptosis and necrosis of the hepatocytes.The apoptosis rate was significantly higher for the high-dose group than for the control (no exposure) group (24.7％±2.39％ vs.4.1％±0.78％,q =8.06,p ＜ 0.05).Expression of the CPS-Ⅱ mRNA was significantly elevated in response to NH4Cl exposure (vs.the control group;F=191.881,p ＜ 0.05).The CPS-Ⅱ mRNA expression level increased with increasing NH4Cl concentration (grey values:1.040±0.045,1.641±0.123,2.285±0.167 and 3.347±0.124,respectively).The CPS-Ⅱ protein expression level was also significantly enhanced in response to the NH4Cl exposures (CPS-Ⅱ protein and internal GAPDH grey value ratios:0.099±0.0130,0.143±0.025,0.161±0.036 and 0.223±0.042,respectively;t=3.825,3.968 and 6.908,p ＜ 0.05).Conclusion CPS-Ⅱ mRNA and protein expression levels become elevated with increase in the NH4Cl concentrations,suggesting that in addition to the urea cycle,CPS-Ⅱ may play an important role in the ammonia metabolism under the condition of hyperammonemia.