Development Of A Hepatoma Cell Line With Reliable Expression Of Drug Metabolizing Enzymes By PiggyBac

Drug safety evaluation is an important part of drug development. Liver acts as a target organ in drug metabolism tixic reaction, playsing a vital important role in drug safety evaluation. Primary hepatocytes as a gold standard, which is widely used in drug screening. However, the application of primary hepatocytes has been greatly limited for the effect of ethics, the genetic background differences of donor, the limitation of sources and the unstable culturing in vitro. So more and more research has focused on the in vitro hepatocyte model. Hep G2 cells was derived from the liver, and still retains many features characteristic of liver cells and can be stable subcultured in vitro, whch is commonly used in toxicity assessment model. But compared with primary hepatocytes, the expression level of drug metabolizing enzymes is low, which limits its application in drug metabolism and toxicity evaluation.This research used high-efficient transposon gene transfer technology conbined with cell monoclonal cluture method to prepare constructing transgene cell lines and stablity expression of drug-metabolizing enzymes CYP3A4 and CYP2C19. Firstly, constructed the recombinant plasmids with CYP3A4 and CYP2C19 gene for co-expression, then transfected it into Hep G2 cells. After the flow cytometry sorting and limiting dilution method, the 21 monoclonal cells were obtained through the cloning-cylinders technology.We have obtained 21 monoclonal cells by the way of flow cytometry, limiting dilution method and cloning ring technology. Subsequently, we selected three monoclonal cells after observation of the fluorescence microscope,and then we identified them by real-time PCR, Western blotting and LC-MS/MS methods from m RNA, protein and activity levels, respectively.,The experimental results show that three resistant clone cells were apparetly higher than wild type cell in m RNA relative quantity of CYP3A4 and CYP2C19. By the way of Western blot, all the resisitant clone cell expressesd successfully drug metabolizing enzymes CYP3A4 and CYP2C19 which those molecular weight were around 48 k Da and 56 k Da. For the study of enzyme activity level, we made the resistant monoclonal cell interact with metabolizing enzymes substrates. The result of higher metabolized rate of 6β-Hydroxytestosterone and 4-Hydroxymephenytoin showed the transgene cell line with better than wild type cells the the resistant cell clones. Ultimately, we obtained Hep G2 cell lines with stable expression of drug-metabolizing enzyme CYP3A4 and CYP2C19.