微流控芯片毛细管电泳法检测细胞内活性氧与细胞凋亡关系  

Simultaneous Determination of Intracellular Reactive Oxygen Species and Apoptosis Signal

作　　者：孙悦[1] 莫满芳[1] 戴卫平[1] 张玉英[2] 梁楚婷[1] 王淑美[1] 

机构地区：[1]广东药学院中药学院,广东广州510006 [2]广东药学院基础学院,广东广州510006

出　　处：《分析测试学报》2013年第8期968-972,共5页Journal of Instrumental Analysis

基　　金：国家自然科学基金项目(81001600)

摘　　要：建立了微流控芯片毛细管电泳激光诱导荧光同时测定细胞内活性氧和凋亡信号的方法。先用AlexaFluor488 annexin V细胞凋亡试剂盒标记细胞凋亡的外翻磷脂酰丝氨酸,再用双氢罗丹明123(DHR123)标记细胞内活性氧,用PBS将细胞调整为终密度1.2×106cells/mL的细胞悬液。细胞群经反复冻融法破碎后,以20 mmol/L硼砂(pH 9.2)作电泳缓冲溶液,分离电压1.2 kV,进样时间60 s,1 min内可完成活性氧和细胞凋亡信号的同时测定。方法简单、快速,细胞内活性氧和DHR123的反应产物(Rh123)在0.5～3μmol/L浓度范围内线性关系良好,相关系数(r)为0.998,检出限(S/N=3)为0.058μmol/L,可用于细胞内活性氧的定量分析。测得HepG2肝癌细胞活性氧含量为0.16μmol/L,被阿霉素诱导凋亡后,细胞内活性氧含量升高至1.77μmol/L。A novel method for simultaneous determintation of intracellular reactive oxygen species and apoptosis signal was developed by microchip capillary electrophoresis with laser induced fluorescence detection. Alexa Fluor^？ 488 annexin V was used to label phosphatidyl serine in outer surface of the ap optosis cells, and dihydrorhodamine 123 （DHR123） was used to convert ROS to the fluorescent rhoda mine 123 （Rh123） intracellularly. The cells were diluted with PBS to a final density of 1.2 x 10^6 cells/ mL, and then crushed by a repetitive freeze thaw method. The supernatant was separated in 1 min u sing 20 mmoL/L borate buffer（pH 9. 2） as buffer medium, with a separation voltage of 1.2 kV and an injection time of 60 s. Under the optimized experimental conditions, the calibration of Pda123 was line ar in the range of 0. 5 -3 μmol/L with a correlation coefficient （r） of O. 998. The limit of detection（S/ N = 3） was 0. 058 μmol/L. The established method was applied in the HepG2 cancer cell, and ROS in ceils was quantified as 0. 16 μmol/L before and 1.77 μmol/L after apoptosis.

关 键 词：微流控芯片 活性氧 细胞凋亡 

分 类 号：O657.8[理学—分析化学]