摘要
AIM: To establish a luciferase reporter cell line that responds dioxin-like chemicals (DLCs) and on this basis to evaluate its characteristics and application in the determination of DLCs.METHODS: A recombinant luciferase reporter plasmid was constructed by inserting dioxin-responsive element (DREs)and MMTV promoter segments into the pGL3-promoter plasmid immediately upstream of the luciferase gene, which was structurally demonstrated by fragment mapping analysis in gel electrophoresis and transfected into the human hepatoma cell line HepG2, both transiently and stably, to identify the inducible expression of luciferase by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The time course,responsive period, sensitivity, structure-inducibility and doseeffect relationships of inducible luciferase expression to DLCs was dynamically observed in HepG2 cells stably transfected by the recombinant vector (HepG2-Luc) and compared with that assayed by ethoxyresorufin-O-deethylase (EROD) in non-transfected HepG2 cells (HepG2-wt).RESULTS: The inducible luciferase expression of HepG2-Luc cells wa s noted in a time-, dose-, and AhR-dependent manner, which peaked at 4 h and then decreased to a stable level at 14 h after TCDD treatment. The responsiveness of HepG2-Luc cells to TCDD induction was decreased with culture time and became undetectable at 10th month of HepG2-Luc cell formation. The fact that luciferase activity induced by 3, 3', 4, 4′-PCB in HepG2-Luc cells was much less than that induced by TCDD suggests a structureinducibility relationship existing among DLCs. Within the concentrations from 3.5× 10-12 to 5× 10-9 mol/L, significant correlations between TCDD doses and EROD activities were observed in both HepG2-luc and HepG2-wt cells. The correlation between TCDD doses from 1.1×10-13 to 1×10-8 mol/L and luciferase activities was also found to be significant in HepG2-luc cells (r=0.997, P＜0.001), but not in their HepG2-wt counterparts. For the comparison of the enzyme responsiveness between cell lines to TCDD, the luciferase sensitivity and reproducibility in HepG2-luc cells were both better than that of EROD in HepG2-wt cells, the former was at 1.1×10-13mol/L and 3.5× 10-12 mol/L, and the coefficients of variation (CV) of the latter was 15-30 % and 22-38 %, respectively.CONCLUSION: The luciferase expression of HepG2-luc cells established in the present study could sensitively respond to the DLCs stimulation and might be a prospective tool for the determination of DLCs.
AIM:To establish a luciferase reporter cell line that responds dioxin-like chemicals (DLCs) and on this basis to evaluate its characteristics and application in the determination of DLCs. METHODS:A recombinant luciferase reporter plasmid was constructed by inserting dioxin-responsive element (DREs) and MMTV promoter segments into the pGL_3-promoter plasmid immediately upstream of the luciferase gene,which was structurally demonstrated by fragment mapping analysis in gel electrophoresis and transfected into the human hepatoma cell line HepG_2,both transiently and stably,to identify the inducible expression of luciferase by 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD).The time course, responsive period,sensitivity,structure-inducibility and dose- effect relationships of inducible luciferase expression to DLCs was dynamically observed in HepG_2 cells stably transfected by the recombinant vector (HepG_2-Luc) and compared with that assayed by ethoxyresorufin-O-deethylase (EROD) in non-transfected HepG_2 cells (HepG_2-wt). RESULTS:The inducible luciferase expression of HepG_2- Luc cells was noted in a time-,dose-,and AhR-dependent manner,which peaked at 4 h and then decreased to a stable level at 14 h after TCDD treatment.The responsiveness of HepG_2-Luc cells to TCDD induction was decreased with culture time and became undetectable at 10^(th) month of HepG_2-Luc cell formation.The fact that luciferase activity induced by 3,3',4,4'-PCB in HepG_2-Luc cells was much less than that induced by TCDD suggests a structure- inducibility relationship existing among DLCs.Within the concentrations from 3.5×10^(-12) to 5×10^(-9) mol/L,significant correlations between TCDD doses and EROD activities were observed in both HepG_2-luc and HepG_2-wt cells.The correlation between TCDD doses from 1.1×10^(-13) to 1×10^(-8) mol/L and luciferase activities was also found to be significant in HepG_2- luc cells (r=0.997,P<0.001),but not in their HepG_2-wt counterparts.For the comparison of the enzyme responsiveness between cell lines to TCDD,the luciferase sensitivity and reproducibility in HepG_2-luc cells were both better than that of EROD in HepG_2-wt cells,the former was at 1.1×10^(-13) mol/L and 3.5×10^(-12) mol/L,and the coefficients of variation (CV) of the latter was 15-30% and 22-38%,respectively. CONCLUSION:The luciferase expression of HepG_2-luc cells established in the present study could sensitively respond to the DLCs stimulation and might be a prospective tool for the determination of DLCs.
基金
the National Natural Science Foundation of China,No.29877020 and 20107002
