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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Importance of culturing primary lymphocytes at physiological oxygen levels
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Importance of culturing primary lymphocytes at physiological oxygen levels

机译：在生理氧气水平下培养原代淋巴细胞的重要性

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Although studies with primary lymphocytes are almost always conducted in CO_2 incubators maintained at atmospheric oxygen levels (atmosO_2; 20%), the physiological oxygen levels (physO_2; 5%) that cells encounter in vivo are 2-4 times lower. We show here that culturing primary T cells at atmosO_2 significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO_2 maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO_2 than at physO_2. This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: (ⅰ) the intracellular NO (iNO) levels are higher at physO_2 than at atmosO_2; and (ⅱ) the peak expression of CD69 is significantly delayed and more sustained at physO_2 that at atmosO_2. Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo, the lower proliferative T cell responses at physO_2 likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO_2 because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.
机译：尽管对原代淋巴细胞的研究几乎总是在维持在大气氧水平（atmosO_2； 20％）的CO_2培养箱中进行，但细胞在体内遇到的生理氧水平（physO_2； 5％）要低2-4倍。我们在这里显示，在atmosO_2上培养原代T细胞会显着改变细胞内的氧化还原状态（降低细胞内的谷胱甘肽，增加氧化的细胞内谷胱甘肽），而在physO_2上的培养则保持细胞内的氧化还原环境（细胞内的谷胱甘肽/氧化的细胞内的谷胱甘肽）接近其体内状态。此外，我们显示，在atmosO_2上，CD3 / CD28诱导的T细胞增殖（基于增殖指数和细胞产量）高于physO_2。我们认为，这一明显矛盾的发现可以用CD3 / CD28刺激的T细胞的另外两个发现来解释：（1）physO_2的细胞内NO（iNO）水平高于atmosO_2的细胞内NO（iNO）水平； （ⅱ）在physO_2处，与atmosO_2处相比，CD69的峰值表达显着延迟并且更持久。因为高水平的细胞内NO和持续的CD69倾向于在体内下调T细胞反应，所以在physO_2处较低的增殖性T细胞反应可能反映了天然体内调节机制的体外操作。因此，我们建议在atmosO_2培养原代淋巴细胞时要谨慎，因为对非生理氧水平的必要适应可能会严重扭曲T细胞反应，尤其是在培养几天后。

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