New strategy for oxidative stress studies with a newly developed device for O_2^- generation

本装置在细胞实验中的应用我们用原装置研究了活性氧对几种培养细胞的影响。当将其应用于HEK 293细胞时，在30小时后发生明显的细胞死亡。当其用于CHO细胞时，细胞生长受到抑制，但在相同浓度下未发生细胞死亡。另一方面，该装置不影响Hela细胞的生长或活力。这些结果表明，活性氧的影响取决于细胞类型和癌细胞是相当抵抗活性氧。在HEK 293细胞的实验中使用SOD或过氧化氢酶，我们发现实际上有效的活性氧是过氧化氢。设备II的开发，原始设备的第二个版本不幸的是，原始设备被发现在细胞培养基中不稳定。因此，我们试图通过化学交联来提高器件的稳定性。用交联剂EDC和磺基-NHS处理稍微稀释的装置。所得器械在培养基（例如MEM）中变得非常稳定。活性的半衰期超过3小时，即使在5小时后，该设备仍显示出40%的活性。装置Ⅱ在氧化应激实验中的应用将该装置应用于人HL-60细胞，检测活性氧对细胞的影响。在用装置II和NADPH孵育22小时后，观察到细胞死亡，细胞数量减少至初始的30%。当添加不含NADPH的器械时，未观察到此类现象。超氧化物歧化酶（SOD）对该效应的影响不大，过氧化氢酶（CAT）对该效应的阻断作用最大，说明实际反应的物种也是过氧化氢。

Application of the device to cell experimentsWe examined the effect of reactive oxygen species on several cultured cells using the original device. When it was applied to HEK293 cells, apparent cell death took place after 30h. When it was used for CHO cells, cell growth was suppressed but cell death did not occur at the same concentration. On the other hand, the device did not affect growth or viability of Hela cells. These results suggested that the effect of reactive oxygen species varies depending on cell types and carcinoma cells were fairly resistant to reactive oxygen species. Using SOD or catalase in the experiments with HEK293 cells we found that reactive oxygen species that is actually effective is hydrogen peroxide.Development of device II, the second version of the original deviceUnfortunately, the original device was found to be unstable in cell culture media. Therefore, we tried to improve the device in stability by chemical cross-linking. The device, somewhat diluted, was treated with a cross-linker EDC and sulfo-NHS. The resulting device became very stable in a culture medium (e.g.MEM). The half-life of the activity was over 3h and the device showed 40% activity even after 5h. The new device was referred to as "device II".Application of device II to oxidative stress experimentsThe new device was applied to human HL-60 cells and examined the effect of reactive oxygen on the cells. After incubation with device II and NADPH at 22h, cell death was observed and the cell number decreased to 30% of the initial. When the device was added without NADPH, such phenomena was not seen. SOD did not influence the effect much, but catalase blocked the effect mostly, indicating that the actual reacting species is also hydrogen peroxide.