流式细胞术检测毕赤酵母发酵过程中胞内活性氧水平

以2′,7′-二氢二氯荧光黄双乙酸钠(DCFH-DA)和碘化丙锭(PI)为标记探针,通过DCFH-DA/PI双染色与PI单染色的对照,检测毕赤酵母胞内活性氧(reactive oxygen species,ROS)的水平及其影响。研究发现发酵过程细胞活性下降与胞内ROS积累相关。在甘油生长期,细胞几乎没有ROS积累,细胞活性接近100%。在甲醇诱导初期,部分细胞积累少量的ROS,细胞活性仍然很高,死亡细胞所占比例只有1.5%。在甲醇诱导后期,94.0%的细胞积累了大量的ROS,高含量的ROS造成细胞损伤,引起部分细胞丧失了活性,在总共29.1%的死亡细胞中,高ROS积累的死亡细胞占了25.4%。

Detection of Intracellular Reactive Oxygen Species by Flow Cytometry in Pichia pastoris Fermentation

In Pichia pastoris fermentation, methanol was oxidized into carbon oxide and produced a byproduct H2 O2, one of the partially reduced forms of molecular oxygen known as reactive oxygen species (ROS) . ROS are highly damaging towards cellular constituents. Flow cytometry (FCM) is an excellent method that permits the rapid, optical analysis of individual cells and has many advantages over conventional cytometry. However, its use in detecting intracellular ROS levels during Pichia fermentation was rarely reported. In our work, by means of flow cytometry, two fluorescent dye 2', 7'-dichlorofluorescin diacetate (DCFH-DA) and propidium iodide (PI) were used to detect ROS. The effect of intracellular ROS on Pichia pastoris cells during fermentation was studied through the comparison between DCFH-DA/PI double-stained cells and PI single-stained cells. In this study, the loss of cell viability during fermentation was correlated with the accumulation of ROS. At the glycerol batch and fed-batch phase, little ROS was accumulated intracellularly and cell viability reached almost 100%. At the early methanol fed-batch phase, intracellular ROS accumulation took place but 98.5% cells still kept viable. At the later methanol fed-batch phase, 94.0% cells accumulated high ROS. As a result, some cells lost their viability because of the damage of ROS. 25.4% dead cells accumulated high ROS in the total 29.1% dead cells.