中文摘要：

【目的】探讨不同的代谢活化系统在人细胞转化模型中潜在的应用价值。【方法】在永生化人肝细胞L02中导入第12密码子突变的H-RAS癌基因（L02-R细胞）,使其处于恶性转化前的易感状态。选择具有代表性的间接致癌物黄曲霉毒素B1（aflatoxin B1,AFB1）作用于永生化和转化前期细胞株,采用三种代谢系统：高表达代谢酶P450 CYP1A2,低剂量底物诱导和加入经典的大鼠S9代谢辅助系统,通过软琼脂克隆形成试验和裸鼠皮下成瘤试验来比较在不同的代谢活化系统下,间接致癌物诱导细胞转化的效能。【结果】蛋白印迹验证H-Ras稳定高表达的转基因细胞株构建成功。通过Ames试验,蛋白印记和CYP1A2酶活性等方法比较三种代谢系统酶活性。在未加代谢系统的条件下,经AFB1染毒后第17周L02-R细胞发生转化,而对照组细胞L02-V在染毒20周未能发生转化。而加入S9代谢系统时,AFB1诱导L02-R细胞在第8周发生转化,比未加代谢系统缩短了9周;经低剂量0.1μmol/LAFB1诱导和高表达CYP1A2均使L02-R的转化间期缩短6周,于第11周发生转化。【结论】三种代谢系统都能够促进间接致癌物AFB1的代谢活化,缩短细胞转化的时间,提高细胞转化效率。与传统的S9代谢活化系统相比,低剂量诱导作为新的代谢活化的方法在细胞转化试验中有着良好的应用前景。

英文摘要：

[ Objective] To study the potential application value of different metabolic activation systems in human cell transformation models. [Methods] L02-R cells were established through introduction of an oncogenic allele of H-RAS V12 into an immortal hepatic cell line L02 and were set in the susceptive condition of premalignant transformation. The immortal L02 and pre- transformation stage L02-R cells were treated with the representative indirect carcinogen aflatoxin B, （AFB1）, under three in vitro metabolic activation systems： overexpression metabolic enzyme P450 CYP1A2, low-dose substrate induction, and the metabolic auxiliary system added with classic rat S9. Through soft agar colony formation assay and subcutaneous tumor fornmtion assay in nude rats, the efficiency of indirect carcinogens inducing cell transformation in different metabolic activation systems was evaluated. [Results] Western blot analysis showed that the H-Ras stable overexpression transgenie cells were established successfully. Through Ames test, Western blot, and detection of CYP1A2 enzyme activity, the enzyme activity of these three metabolic activation systems were compared. Without an additional metabolic system, AFB, exposed L02-R cells transformed at the 17th week, while control cells LO2-V could not be transformed in 20 week after AFBI treatment. When S9 metabolic system was added, AFB, inducing L02-R cells transformed at the 8th week which was 9 weeks shorter than that of adding no metabolic systems. Low-dose 0.1 μmol/L AFB1 induction and overexpression CYP1A2 could shorten the transformation latency of L02-R for 6 week and L02-R transformed at the l lth week. [Conclusions] The three metabolic activation systems can promote the metabolic activation of indirect carcinogens AFB1, shorten the transformation latency, and increase the transformation efficiency. The low-dose chemical induction seems to be a prospective metabolic activation system used in cell transformation assay compared with the traditional S9 metabolic activation syste