Korystov Yuri N, Shaposhnikova Vera V, Korystova Antonina F, Emel'yanov Maksim O
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Moscow Region, Russia.
Radiat Res. 2007 Aug;168(2):226-32. doi: 10.1667/RR0925.1.
The goal of this study was to determine the amount of reactive oxygen species (ROS) that arises inside cells irradiated in medium containing blood serum using the 2'7'-dichlorofluorescein (DCF) assay. DCF fluorescence in cells and medium was recorded on an MF44 Perkin Elmer fluorimeter, and fluorescence in cells only was recorded on a Partec flow-through cytometer. Human larynx tumor HEp-2 cells and lympholeukosis P388 cells were irradiated with X rays at a dose rate of 1.12 Gy/min. The factors (temperature, pH, serum concentration) affecting the oxidation of 2'7'-dichlorofluorescin (DCFH) to DCF were studied, and errors in the dichlorofluorescein assay of ROS were minimized. The amount of ROS registered by the DCF assay in cells was found to depend on the concentration of serum in the medium during irradiation. In the presence of 10% serum, radiation had no effect on the amount of detectable ROS. The effect of radiation on the formation of intracellular ROS was almost completely abolished if the irradiated medium was removed immediately after radiation exposure. The increase in the formation of ROS in cells irradiated in medium with a low serum content is due mainly to the radiolytic products of water that arise in medium and oxidize DCFH located in cells.
本研究的目的是使用2'7'-二氯荧光素（DCF）测定法，确定在含有血清的培养基中受照射细胞内产生的活性氧（ROS）的量。细胞和培养基中的DCF荧光在MF44珀金埃尔默荧光计上记录，仅细胞中的荧光在帕尔特流式细胞仪上记录。人喉肿瘤HEp-2细胞和淋巴细胞白血病P388细胞以1.12 Gy/min的剂量率接受X射线照射。研究了影响2'7'-二氯荧光素（DCFH）氧化为DCF的因素（温度、pH值、血清浓度），并将ROS二氯荧光素测定中的误差降至最低。发现通过DCF测定法在细胞中检测到的ROS量取决于照射期间培养基中血清的浓度。在存在10%血清的情况下，辐射对可检测到的ROS量没有影响。如果在辐射暴露后立即去除受照射的培养基，辐射对细胞内ROS形成的影响几乎完全消除。在低血清含量的培养基中受照射的细胞中ROS形成的增加主要是由于培养基中产生的水的辐射分解产物，这些产物氧化位于细胞内的DCFH。
