转基因细胞系CHL-3A4的建立及其代谢活化作用

Establishment of Transgenic Cell Line CHL 3A4 and Its Metabolic Activation

目的建立ＣＨＬ３Ａ４细胞系为某些遗传毒性化合物的代谢途径研究提供模型细胞系，并用于新合成化学物质的致突变性检测。方法应用ＲＴＰＣＲ和ＤＮＡ重组技术从人肝组织中克隆细胞色素Ｐ４５０３Ａ４基因的ｃＤＮＡ至ＢｌｕｅｓｃｒｉｐｔＭ１３载体上，经限制性内切酶图谱分析和克隆片段部分序列测定，证实为ＣＹＰ３Ａ４ｃＤＮＡ。然后构建真核细胞重组表达质粒ｐＲＥＰ９３Ａ４，导入中国仓鼠ＣＨＬ细胞。结果建立了ＣＨＬ３Ａ４转基因细胞系，双核细胞微核试验证明该细胞系能代谢活化黄曲霉毒素Ｂ１（ＡＦＢ１）、杂色霉菌毒素（ＳＴＣ）、环磷酰胺（ＣＰＡ）。结论所建ＣＨＬ３Ａ４细胞系确实能表达人细胞色素Ｐ４５０３Ａ４，并能代谢活化ＡＦＢ１、ＳＴＣ、ＣＰＡ。

Objective To establish a model cell line CHL 3A4 to study metabolic pathways for some genotoxic chemicals. Methods Complimentary DNA (cDNA) of cloned cytochrome P450 3A4 gene in human liver tissue was transferred to Bluescript M13 vector with reverse transcription polymerase chain reaction (RT PCR) and DNA recombinant techniques. Restriction endonuclease map analysis and sequencing of partial cloned fragment proved that CYP3A4 cDNA was cloned into Bluescript M13 vector. Then, recombinant expression plasmid pREP9 3A4 was constructed in eukaryotic cell and transfected into Chinese hamster CHL cells. Results It was proved that a CHL 3A4 transgenic cell line was established, which could lead metabolic activation for aflatoxin B1 (AFB1), sterigmatocystin (STC) and cyclophosphamide (CPA). Conclusion The CHL 3A4 cell line established did express human cytochrome P450 3A4 and could lead metabolic activation for three genotoxic chemicals AFB1, STC and CPA.