Effects Of Recovery Of Two Ammonia Metabolism Pathways On The Biological Functions Of HepG2Cells

AIM： HepG2is an immortalized human hepatoma cell line that has been used tobioartificial liver research. However, the insufficient capacity of ammoniadetoxification limit its further application due to absent or low expression of hGS,hArgI and hOTC enzyme in the two ammonia metabolism pathways. On the basis ofthe previously established HepG2/(hArgI+hOTC)4cells with over-expression ofhArgI and hOTC, we employed the recombinant retroviruses, Retro-AFhGS, to infectHepG2/(hArgI+hOTC)4cells and try to recovery the two ammonia metabolismpathways of HepG2cells in this work.METHODS: The eukaryotic expression vectors, pLNCX and pLNAFhGS, weretransfected into HepG2/con and HepG2/(hArgI+hOTC)4(previously established)cells, and then the G418-resistant cell clones, HepG2/Mock andHepG2/(hArgI+hOTC+AFhGS)1, were selected and amplified for verificationexperiments. The ammonia metabolism activity and cell proliferation were evaluatedby CCK-8kit, enzymatic activity of hArgI and hOTC, and synthesis of urea weremeasured by specific biochemical methods, and glutamine was detected using aquantitative biochemistry kit. The protein expression of hArgI, hOTC, hCPSI, hASS,hASL and hArgII were testified by Western-blot. Finally, the ammonia metabolismactivities and their possible mechanism of these three recombinant HepG2cells werefurther evaluated. Moreover, the effect of hGS transduction on HepG2proliferationwas analyzed by cell cycle PCR chip, and the related change genes were testified byRT-PCR.RESULTS: The hGS gene was successfully transducted into HepG2/(hArgI+hOTC)4cells. The ammonia tolerance capacity of HepG2/AFhGS, HepG2/(hArgI+hOTC)4and HepG2/(hArgI+hOTC+AFhGS)1cells was higher than HepG2and HepG2/Mockcells. The amount of urea and glutamine synthesis of HepG2/AFhGS,HepG2/(hArgI+hOTC)4and HepG2/(hArgI+hOTC+AFhGS)1cells at higher ammonia concentrations were more than that of HepG2and HepG2/Mock (P<0.05).The hArgI expression and enzymatic activity decreased in HepG2/AFhGS andHepG2/(hArgI+hOTC+AFhGS)1cells, the hOTC expression and enzymatic activityincreased in HepG2/(hArgI+hOTC)4and HepG2/(hArgI+hOTC+AFhGS)1cells, andthe expression of hASL, one of other three key enzymes in urea cycle, was higher inHepG2/AFhGS and HepG2/(hArgI+hOTC)4cells (P<0.05). In addition, thetransduction of hGS promoted hArgII expression in HepG2/AFhGS andHepG2/(hArgI+hOTC+AFhGS)1cells to a certain extent. HepG2/AFhGS cells growslowerly at the fifth and seventh day than other cells (P<0.05), and the proliferation ofHepG2, HepG2/Mock, HepG2/(hArgI+hOTC)4and HepG2/(hArgI+hOTC+AFhGS)1cells had no significant difference(P>0.05). The difference of hepatic basic functionalparameters of these cells was not significant (P>0.05), except the albumin synthesisof HepG2/(hArgI+hOTC)4cell higher than other four cells (P<0.05). Gene chipresults showed that the CDC16and HERC5expression were increased, and the BCL2expression was decreased significantly in HepG2/AFhGS cells, but only CDC16expression was up-regulated in HepG2/(hArgI+hOTC)4cells.CONCLUSION: The established HepG2/(hArgI+hOTC+AFhGS)1cell had a lessability to degrade ammonia in vitro compared with HepG2/AFhGS andHepG2/(hArgI+hOTC)4cells. The transduction of hGS gene could inhibit the hArgIexpression of HepG2/(hArgI+hOTC)4. The decreased proliferation of HepG2/AFhGScell was due to the down-regulated expression of anti-apoptosis gene BCL2. Insummary, the HepG2/(hArgI+hOTC)4cells are the most suitable for the futher BALapplication study in animal with hepatic failure.