Construction And Function Evaluation Of Liver Cell Line Tranfected With Vector Expressing Cytochrome P450 3A4 And Glutathione-S-transferase A1

The hepatic metabolism of drug decreases obviously in patients with serious liver diseases.This reduced the drug effects and aggravated the liver damage, which is due to the declined expression and activity of drug metabolic enzyme. Therefore, we have targeted gene cytochrome P450 3A4 (CYP 3A4) and glutathione-S-transferase A1 (GST A1), which belonged to PhaseⅠand PhaseⅡdrug metabolic enzyme respectively. At the same time, we optimized the codons of CYP 3A4 and GST A1 to enhance the expression. The two vectors transfected C3A cell line and the function of drug metabolism was evaluated.1. Construction and identification of recombinant plasmids The optimized CYP 3A4 and GST A1 were synthesized and cloned to pcDNA3.1(+) which had two CMV promoters. The recombinant plasmid was named pcDNA3.1(+)-OCYP 3A4-OGST A1. And we have gotten the OCYP 3A4 and OGST A1 gene by polymerase chain reaction(PCR) from this plasmid. By means of digestion, ligation and transformation, we got two recombinant plasmids, named pBudCE4.1-CYP 3A4-GST A1 and pBudCE4.1-OCYP 3A4-OGST A1 respectively, and confirmed by sequencing and blast analysis.2. Establishment and function evaluation of transfected liver cell line C3A cell line were transfected with recombinant plasmids of pBudCE4.1-CYP 3A4-GST Al and pBudCE4.1-OCYP 3A4-OGST A1 respectively, and cultured with MEM containing 400μg/ml Zeocin for 2 weeks. The cell lines, named C3A-Unoptimized, expressed CYP 3A4 and GST A1 successfully and stably, while the cell lines expressed OCYP 3A4 and OGST Al unstably. By MTT assay, it showed that the cell growth would be inhibited by the increased expression of CYP 3A4 and GSTAl. The C3A-Unoptimized cell lines expressed more CYP 3A4 and GSTA1 than that of normal C3A cell line tested by qPCR. By chromatogram assay, it showed that the activity of CYP 3A4 existed in the C3A-Unoptimized cell lines whereas it was undetectable in normal C3A cell line. The immuno-histochemical staining indicated the higher expression of GSTA1 in C3A-Unoptimized cell lines than in normal C3A cell line. The ability of metabolizing lidocarine for the C3A-Unoptimized cell lines was enhanced (the metabolizing rate was 62.5%) comparing with that in normal C3A cell line (the metabolizing rate was 30%). Conclusions:The function of C3A-Unoptimized cell line have been improved, and this cell line might become a new cell material in the bioartificial liver support system.