| Summary: | 碩士 === 長庚大學 === 醫學生物技術暨檢驗學系 === 99 === Glucose-6-phosphate dehydrogenase (G6PD) is an important enzyme for maintaing redox homeostasis, and can produce NADPH to scavenge reactive oxygen species (ROS). In our study, we increased oxidative stress by adding Glucose oxidase (GOx), which utilized β-D-glucose to produce H2O2, into the cell culture medium. By using this strategy, we analyzed the effects of GOx on G6PD-knockdown Hep-G2 and A549 cells. First, we added GOx to cell-free medium, and found that the H2O2 concentration was increased in a dose-dependent manner. We further determined the concentration of H2O2 in culture medium, after adding GOx to G6PD-knockdown and control cells. No significant differences in H2O2 concentration generated by GOx in medium between Hep-G2 and A549 cells. Furthermore, we added GOx at 10 and 17.5 mU/ml to HepG2 and A549 cells, respectively, and found that the viability of G6PD-knockdown HepG2 and A549 cells was lower than control cells. We also discovered that there were a 3-fold increase in SubG1 phase in G6PD-knockdown cells comparing to control cells following GOx treatment. We then used this GOx-mediated H2O2 generation system to determine the protective effect of some antioxidants such as N-acetylcysteine (NAC), Pistacia weinmannifolia (PW), Lipoic acid, as well as other antioxidants. The results indicate that NAC, PW, and Lipoic acid could ameliorate GOx-induced HepG2 cell death in this system, while the other antioxidants had little or no effect. Taken together, this study indicates that the H2O2 generating system by GOx is a good system to demonstrate G6PD-deficient cells are more susceptible to oxidants.
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