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

使用新开发的 O_2^- 生成装置进行氧化应激研究的新策略

基本信息

批准号：
15300164
负责人：
TAMURA Minoru
金额：
$ 9.73万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15300164/
关键词：
Superoxide Reactive oxygen intermediate NADPH oxidase Cytochrome b_<558>Fusion protein Oxidative stress Cell culture Molecular device シトクロム b_<558>スーパーオキサイド NADOH oxidase タンパク融合 Superoxide ROS Cytochrome b_<558>Fusion prot

项目摘要

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.

该装置在细胞实验中的应用我们使用原始装置检测了活性氧对几种培养细胞的影响。作用于HEK293细胞，30h后细胞明显死亡。当它用于CHO细胞时，在相同浓度下细胞生长受到抑制，但细胞死亡未发生。另一方面，该装置不影响海拉细胞的生长或活力。这些结果表明，活性氧的作用因细胞类型而异，而癌细胞对活性氧具有相当的抗性。在HEK293细胞实验中使用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，原设备的第二版不幸的是，原设备被发现在细胞培养基中不稳定。因此，我们尝试通过化学交联来提高器件的稳定性。将该装置稍微稀释后，用交联剂EDC和磺胺- nhs处理。该装置在培养基（如mem）中变得非常稳定。活性半衰期超过3h， 5h后仍有40%的活性。这种新设备被称为“设备II”。装置II在氧化应激实验中的应用将新装置应用于人HL-60细胞，检测活性氧对细胞的影响。用装置II和NADPH孵育22h后，观察到细胞死亡，细胞数量减少到初始的30%。在不添加NADPH的情况下，没有出现这种现象。SOD对效果影响不大，过氧化氢酶对效果影响最大，说明实际反应的物质也是过氧化氢。