Establishment of a Gastirc Carcinoma Cell Line for Reporter Gene Assay of Dioxin-Like Chemicals

摘要

For the aim of quick and convenient detection of dioxin-like chemicals molecules and understanding the function of dioxin response element in human cell line, we developed a cellular model in which aryl hydrocarbon receptor activities could be indicated by EGFP (enhanced green fluorescence protein). Firstly, a five copies XRE (xenobiotic response element) fragments and minimal CMV (Human cytomegalovirus) promoter were inserted into pEGFP-1 to generate a new vector-pXRE5-EGFP. This vector contains the EGFP gene under TCDD-inducible control of five dioxin-responsive DNA enhancer elements. This reporter gene system is based on human gastric carcinoma cell stably transfected with the vector pXRE5-EGFP which allows expression of the EGFP under control of the dioxin regulatory element of the human CYP 1A1 gene. A stably transfected cell line XRE5-SGC-7901 was established and cloned under pressure of selective antibiotic G-418. This in vitro model allowed the study of an aryl hydrocarbon receptor activity by the detection of the induced EGFP. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) induced the EGFP activity in a dose-dependent manner. The induced fluorescence intensity measured by flow cytometry reached a maximum level at 48 hours after the cells were incubated with 500 pg/mL TCDD.