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目的 研究 3 甲基胆蒽 (3 methylcholanthrene,3 MC)对原代培养大鼠肝细胞细胞色素P450 1A(CYP 1A)酶活力的诱导作用 ,并探讨其时间 效应和剂量 效应关系。方法 原位 2步胶原酶灌流法分离大鼠肝细胞 ,无血清条件下培养细胞 ,并用不同剂量的 3 MC诱导肝细胞 2 4h或 48h。乙氧基试卤灵 (ethoxyresorufin,EOR)为CYP 1A酶活力探针药 ,高效液相色谱法 (HPLC)测定试卤灵 (resorufin,RSF)浓度。结果 实验条件下对照组和 3 MC诱导组的RSF均可被检测 ,与对照细胞相比 ,除 0 0 0 5μmol L的 3 MC对原代培养大鼠肝细胞EROD无显著诱导作用外 ,其余剂量 (0 0 5 ,0 5 ,5μmol L)的 3 MC均明显诱导酶活力 ,酶活力分别被上调至对照组的 5 0 8,2 3 3 ,33 6倍 (P <0 0 1 ) ,3 MC的诱导作用在 2 4h达最强 ,而其在 48h的诱导作用与 2 4h相似。3 MC对原代培养大鼠肝细胞CYP 1A酶活力的诱导作用呈明显的剂量依赖性。结论 3 MC对大鼠原代培养肝细胞CYP 1A酶活力有明显的诱导作用
Objective To investigate the induction of 3-methylcholanthrene (3 MC) on the activity of cytochrome P450 1A (CYP 1A) in primary cultured rat hepatocytes and investigate the relationship between time effect and dose-response. Methods In situ 2-step collagenase perfusion method was used to separate rat hepatocytes. Cells were cultured under serum-free conditions. Hepatocytes were induced with 3 MC at different doses for 24 h or 48 h. Ethoxyresorufin (EOR) was a CYP 1A enzyme probe and the concentration of resorufin (RSF) was determined by high performance liquid chromatography (HPLC). Results Under the experimental conditions, the RSFs of control group and 3 MC-induced group were all detected. Compared with control cells, no significant effect of 3 0 MC of 0 0 0 5 μmol L on primary cultured rat hepatocytes EROD was observed. (0 0 5, 0 5, 5μmol L) 3 MC significantly induced enzyme activity, enzyme activity were up-regulated to 50.8, 23.3, 33.6 times (P <0.01), 3 MC Induced at 24 h was the strongest, while its induction at 48 h was similar to 24 h. 3 MC induced primary cultured rat liver CYP 1A enzyme activity in a significant dose-dependent manner. Conclusions 3 MC can obviously induce CYP 1A enzyme activity in rat primary cultured hepatocytes