| Objective:In this study,we separately constructed lentivirus that contain the liver enrichment transcription factor and the allogeneic nuclear receptor,and screened the optimal combination of LETF and heterokaryon nuclear receptors that have an significant effect on ammonia metabolism?urea circulation pathways?and drug metabolism in hepatocytes.Finally,we constructed hepatocytes that could be used simultaneously for BAL and hepatotoxicity detection.Methods:1.Using adenovirus strategy,several common LETFs?C/EBP?,HNF3?,HNF4?,HNF1?,HNF6 and PGC1??were multiple combination screened for their transient expression characteristics.We detected the mRNA expression levels of five key enzymes of urea cycle?Arg1,OTC,CPS1,ASS and ASL?by real-time quantitative PCR,and then screened out five kinds of urea cycle key enzyme positive regulation of liver enrichment transcription factor combination.2.Based on this combination,nuclear receptors?CAR and PXR?,which were involved in the regulation of CYP enzyme synthesis were added and multiple combinations were screened again.Real-time quantitative PCR was used to detect the mRNA expression levels of five key enzymes of urea cycle and five cytochrome oxidase P450s?CYP1A1,CYP2B6,CYP29,CYP2D6 and CYP3A4?and using WB to detect the level of protein synthesis at the same time.3.Due to the stable expression of lentivirus,we constructed a hepatocyte cell line overexpressing LETFs and heterologous nuclear receptors.4.The production of hepatic urea was tested to validate the metabolic function of urea in recombinant hepatocyte cell line.At the same time,the CYP function of recombinant hepatocyte cell line was detected indirectly by CYP enzyme kit.Ultimately the effects of optimized combination of LETFs and allogeneic nuclear receptors was determined on hepatocyte function.Results:1.Previous studies on Arg1,CPS1,OTC three key enzyme promoters,combined with the existing literature,obtained hepatic enrichment transcription factors C/EBP?,HNF3?,HNF4?,HNF1?,HNF6 and PGC1?which is closely related to transcriptional regulation and heterotopic nuclear receptors PXR and CAR,and constructed in adenovirus respectively.2.34 different combinations of transcription factor adenovirus were instantly transfected into HepG2 cells,and real-time fluorescence quantitative detected mRNA expression of five key enzymes of urea?results with 2-??Ct??Ct value?.The results showed that under the combined effect of different transcription factor combinations,the expression of five kinds of urease were different.Comprehensive considerations,the use of exclusion method,After preliminary screening to exclude some obviously inappropriate combinations,the actual qPCR expression values of the five enzymes in the remaining combinations were analyzed,and three suitable combinations were selected for the next experiments,namely"C/EBP?+HNF3?+HNF4?","C/EBP?+HNF3?+HNF1?","C/EBP?+HNF4?+HNF6".3.In order to take full account of CYP enzyme expression,the combination of the above three transcription factors adenovirus and nuclear receptors PXR and CAR were 11 combinations,of these 11 new combinations were re-transfected HepG2cells respectively.Real-time fluorescence quantitative detected mRNA expression of five urea metabolizing enzymes and five CYP enzymes?CYP1A1,CYP2B6,CYP2C9,CYP2D6 and CYP3A4?.The results showed that under the combined action of different transcription factors,the expression of ten kinds of functional enzymes were different.For a more intuitive result,all 2-??Ct results were segmented with"<1","1-2",">2"and labeled with different colors,The color depth and mRNA expression level is proportional to the relationship.After comprehensive analysis,two combinations of"C/EBP?+HNF4?+HNF6+PXR"and"C/EBP?+HNF4?+HNF6+CAR"were selected and constructed HepG2 cells and BEL-7404 cells which overexpress two kinds of optimal combination stably.4.WB results showed that ASL protein was highly expressed in the recombinant cells of two different combinations,while the ASS protein was in a low level.In addition,Arg1,OTC and CPS1 showed high expression under the combination of C/EBP?+HNF4?+HNF6+PXR.5.The amount of urea synthesis of the two recombinant cells was further examined to assess the metabolic capacity of the recombinant cells for ammonia.With the increase of ammonia concentration,the urea production of two different combinations of recombinant cells increased to some extent.Moreover,C/EBP?+HNF4?+HNF6+PXR combination was significantly higher than that of control group and C/EBP?+HNF4?+HNF6+CAR combination at low ammonia concentration.In the condition of high concentration of ammonia,urea production of C/EBP?+HNF4?+HNF6+CAR recombinant hepatocytes increased to a certain extent.However,the metabolism of urea in two different combinations of hepatocytes is somewhat less advanced than in primary hepatocytes.6.Simultaneous detection of five different CYP enzyme protein synthesis levels and functions in two recombinant cells.Under the combination of two different transcription factors,the expression of CYP1A1,CYP2D6 and CYP3A4 protein was significantly increased,whereas the expression of CYP2B6 protein was low and the expression of CYP2C9 was not changed.This effect is further validated in functional assays.Conclusion:1.With urea cycle and drug metabolism function analysis,the combination of C/EBP?+HNFF4?+HNF6+PXR and C/EBP?+HNFF4?+HNF6+CAR is ideal.In a certain degree,both combinations can significantly improve the two functions of ammonia metabolism and drug metabolism.2.According to the analysis of urea metabolism function,the combination of C/EBP?+HNFF4?+HNF6+PXRissuperiortothatof C/EBP?+HNFF4?+HNF6+CAR.3.According to the analysis of drug metabolism,the combination of C/EBP?+HNFF4?+HNF6+PXRandthecombinationof C/EBP?+HNFF4?+HNF6+CAR show no significant difference between the two groups.4.Comprehensive analysis,the combination of C/EBP?+HNFF4?+HNF6+PXR can improve both ammonia metabolism and drug metabolism in the meantime and it is expected to provide convenient human liver cells for futher experimental applications. |
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