Study On The Metabolic Activation System Based On The Co-culture System Of Human Hepatocytes

Objective:By constructing two-dimensional co-culture system of human hepatocyte with different combinations,comparing the superiority of different co-culture system as metabolic activation system from the level of cell activity and main cytochrome enzyme gene expression.The metabolic activation ability of dominant co-culture system was preliminarily verified by in vitro micronucleus test,which provided theoretical basis and technical support for the application of humanized cell co-culture metabolic activation system in genotoxicity test and mutagenicity test.Methods1.Construction of co-culture model and observation and determination of cell growth stateMake 6 hole co-culture plates by perforation method.Human hepatoma HepG2/C3A cells,human colorectal adenocarcinoma Caco-2 cells,human tubular epithelial HK2 cells,human umbilical vein endothelial HUVEC cells,human hepatic stellate LX-2 cells were selected as the cell lines for the co-culture model.Single culture group and four groups of two-cell co-culture systems including HepG2/C3A-Caco2(CO),HepG2/C3A-HK2(CH),HepG2/C3A-HUVEC(CU),HepG2/C3A-LX2(CL)and six groups of three-cell co-culture systems including HepG2/C3A-Caco2-HK2(COH),HepG2/C3A-Caco2-HUVEC(COU),HepG2/C3A-Caco2-LX2(COL),HepG2/C3A-HK2-HUVEC(CHU),HepG2/C3A-HK2-LX2(CHL)and HepG2/C3A-HUVEC-LX2(CLU)were set up respectively.Three repeats were applied per group.After the connection of culture medium,cells were cultured for 4 days,and the cell growth was observed.The CCK-8 method was used to measure the growth curve.The administration of single culture group and co-culture group was consistent.2.Gene expression of major metabolic activation-related enzymes in HepG2/C3A cells.The cells in co-culture and single-culture systems were cultured for 24 h.The lysate was added to extract the RNA.RT-qPCR were applied to measure the expression of major phase Ⅰ metabolic enzyme CYP3A4,CYP2E1,CYPIA2,CYP2B6,CYP2C9 and major phase Ⅱ metabolic enzyme GSTA1/2 and UGT1A1 of HepG2/C3A cells in different culture models.3.Preliminary verification of the dominant co-culture metabolic activation model by micronucleus test.A co-culture model with good cell growth and elevated gene expression level p450 metabolic enzymes was selected as the metabolic activation system.The metabolic activation efficacy of the model was validated by in vitro cytokinesis-block micronucleus test.The cyclophosphamide(9.5,19 and 38μg/ml)which a typically mutagen required metabolic activation,and the methanesulfonate(0.325,0.65 and 1.3μg/ml),a positive substance without metabolic activation,were used with blank control and solvent(ultrapure water and dmso)control groups.The test operation of the control group was consistent with that of the experimental group.After the drops were stained with Giemsa staining,the number of micronucleus cells of 1000 binuclear cells was counted.Results1.Construction of co-culture model and observation and determination of cell growth stateAfter 24 h inoculation,cells grew well and the culture medium was connected for each wells.The cell growth condition was good during 4 days.The cells morphology both of the co-culture and the single culture models was observed by microscope,and no significant changes were found.Growth curves of cells were measured by CCK-8 method.The cell growth inhibition not appeared in the two-cell co-culture models except HK2 cells in the HepG2/C3A-HK2 co-culture model.No significant cell growth difference was observed between co-culture model and single culture model in other three two-cell co-culture models on the second or fourth day of inoculation(P>0.05).There was no significant difference in cell growth between single culture cells and the three-cell co-culture models except in the HepG2/C3A-HUVEC-LX2 co-culture model(P>0.05).In the other cultures,on the third or fourth day of inoculation,some cells in the co-culture model were more active than those in the single culture model(,P<0.05).2.Gene expression of major metabolic activation-related enzymes in HepG2/C3A cells.The expression level of phase I metabolic enzyme genes varied greatly in different models.Expression of CYP1A2 gene in the HepG2/C3A-HUVEC,HepG2/C3A-Caco2-HUVEC and HepG2/C3A-Caco2-LX2 co-culture groups was higher than that in the single culture group,which was 6.54,2.34 and 3.42 times,respectively.The difference was statistically significant(P<0.05).The expression of C.YP2B6 gene was increased in the HepG2/C3A-HUVEC co-culture group,which was 1.47 times higher than that in the single culture group,the difference was statistically significant(P<0.05).The expression of CYP2C9 gene in the HepG2/C3A-Caco2-LX2 co-culture group was higher than that in the single culture group,which was 4.2 times higher than that in the single culture group,the difference was statistically significant(P<0.05).Expression of C.YP2E1 gene in the HepG2/C3A-Caco2,HepG2/C3A-HK2,HepG2/C3A-LX2,HepG2/C3A-HUVEC and HepG2/C3A-HUVEC-LX2 co-culture groups was higher than that in the single culture group,which was 1.63,1.33,1.85,1.62,3.4 times,respectively,the difference was statistically significant(P<0.05).The expression of CYP3A4 gene in the HepG2/C3A-Caco2-LX2 co-culture group was higher than that in the single culture group,2.88 times higher than that in the single culture group,the difference was statistically significant(P<0.05).Expression of UGT1A1 genes in the HepG2/C3A-HK2,HepG2/C3A-Caco2-HUVEC and HepG2/C3A-Caco2-LX2 co-culture groups was higher than that in the single culture group,which was 2.27,4.40,4.75 times higher than that in the single culture group,the difference was statistically significant(P<0.05).The expression of GSTA1A2 gene in the HepG2/C3A-Caco2-LX2 co-culture group was higher than that in the single culture group,which was 5.11 times higher than that in the single culture group,and the difference was statistically significant,(P<0.05).3.Preliminary verification of the dominant co-culture metabolic activation model HepG2/C3A-Caco2-LX2 by micronucleus test.Ethyl mesylate,a mutagenogenic positive that does not require metabolic activation,was tested by micronucleus test.The micronucleus rate of single culture group was 16‰,15‰,16‰ respectively,and the micronucleus rate of co-culture group was 14‰,16‰,15‰.No significant difference was found between the single culture group and co-culture group in micronucleus rates(P>0.05).Cyclophosphamide,a mutagenic positive requiring metabolic activation,was also tested by micronucleus test.The micronucleus rate(21‰,18‰,16‰)in the co-culture group was significantly higher than that in the single culture group(12‰,13‰,12‰),and the difference was statistically significant(P<0.05).Conclusion1.The effects of two-cell co-culture on the growth status of the cells in the model were relatively small.In the three-cell co-culture models of HepG2/C3A-HK2-Caco2 and the HepG2/C3A-Caco2-LX2,the cell growth activity was better than that of the single culture model,and the growth was stable,which was expected to become a stable co-culture system.2.The main phase I and phase II metabolic enzyme genes expression of the HepG2/C3A liver cells in the HepG2/C3A-Caco2-LX2 co-culture model was increased.3.The mutagenicity of positive precursor cyclophosphamide was verified by HepG2/C3A-Caco2-LX2 co-culture model as metabolic activation system without adding S9.