RESUMEN
The intracellular hepatitis B surface antigen (HBsAg) content per cell was increased by 7.2-fold in the culture with 1.5% dimethyl sulfoxide (DMSO) compared with that in the control without DMSO, while the extracellular HBsAg production and specific productivity were only improved by 70% and 3.2-fold, respectively. Electron microscope has been employed to reveal large dilated structures within recombinant CHO cells in the presence DMSO. The dilated structures have a distribution within whole cytoplasm, and some dilated areas were engulfed in the nucleus. These large, dilated structures were not observed in the control. Immunogold labeling was used to discover the accumulated HBsAg was localized within these dilated areas, and some HBsAg-specific labels were detected in the nucleus membrane, owing to the encroachment of the dilated areas upon nucleus. The result could help to reveal the mechanism of intracellular HBsAg accumulation in the presence of DMSO.
RESUMEN
The toxic effect of ammonia on rCHO-GS cell decreased obviously due to the transfection of GS system in serum-free culture. The maximum cell density, 15.6 x 10(5) cells/mL was obtained in the culture with 1.42 mmol/L ammonia. The growth of rCHO-GS cell was inhibited with an increased ammonia concentration. However, a cell density of 8.9 x 10(5) cells/mL was obtained when the concentration of ammonia was 12.65mmol/L. The intracellar metabolic pathways were affected due to the decrease of the toxic effect of ammonia on rCHO-GS cell. With the increase of initial ammonia concentration from 0.36mmol/L to 12.65mmol/L, the yield coefficients of cell to glucose and lactate to glucose decreased. The activities of hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) increased by 43%, 140% and 25%, respectively, indicating that the utilization of glucose increased and the glycolysis pathway was more prone to efficient energy metabolism pathway. An increased activity of glutamate-pyruvate aminotransferase (GPT) showed that the conversation from glutamate to alpha-ketoglutarate was shifted to glutamate-pyruvate transamination pathway. The deamination pathway was inhibited due to a decreased activity of glutamate dehydrogenase. In addition, the number of cells in G0/G1 phase increased and the specific production rate of recombinant protein increased by 2.1-fold with the increase of initial ammonia concentration from 0.36mmol/L to 12.65mmol/L.