真菌毒素(DON、 AFB1)诱导HepG2/C3A 细胞凋亡研究
首发时间:2015-09-06
摘要:脱氧雪腐镰刀菌烯醇(DON)和黄曲霉毒素B1(AFB1)是一类常见的毒素类污染物,常共存于小麦、玉米等食品及制品中。采用肝癌细胞HepG2/C3A来研究它们之间的联合毒性以及两者引起细胞凋亡机制。实验采用不同浓度的DON(0.56、1.125、2.25、4.5、6.75 μmol/L)和AFB1(2.5、5、10、20、30 μmol/L)单独及联合作用于HepG2/C3A 细胞24 h后,分别测定细胞增殖抑制率、DNA含量、活性氧含量、以及线粒体膜通透性变化,分析两毒素对细胞的联合作用类型,并通过流式细胞仪测定低浓度条件下细胞凋亡率和细胞周期,采用RTq-PCR测定凋亡相关基因caspase-3、Bax以及Bcl-2 mRNA的相对含量。结果显示,两种毒素对HepG2/C3A细胞混合毒性效应均高于单独作用,经分析后发现两者联合作用类型为加和作用。低浓度作用下均能引起细胞发生凋亡,使得细胞阻滞在S期或者S期和G2/M期,并且引起caspase-3和Bax表达量增加,Bcl-2则下降,从而引发细胞凋亡。
关键词: 脱氧雪腐镰刀菌烯醇 黄曲霉毒素B1 细胞凋亡 联合毒性
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Apoptosis Induced By Deoxynivalenol and Aflatoxin B1 in HepG2/C3A Cells
Abstract:Deoxynivalenol (DON) and Aflatoxin B1 are the most prevalent mycotoxins in cereal-based ingredients or foods and they frequently coexist. To study the toxic effect and mechanism of apoptosis of DON and AFB1, HepG2/C3A cell treated with different concentrations of DON(0.56、1.125、2.25、4,5、6.75 μmol/L) and AFB1(2.5、5、10、20、30 μmol/L) for 24 h was used to analyze their toxic effects expressed by cell proliferation inhibition rate (SRB), total double-stranded DNA, intracellular reactive oxygen species (ROS) and mitochondrial membrane permeability (MMP). The apoptosis rate and cell cycle arrest were also studied with lowest concentrations of DON (0.56 μmol/L) and AFB1 (2.5 μmol/L) and their combination and apoptosis-related gene, caspase-3, Bax, Bcl-2 were analyzed by real-time quantitative PCR .The results showed that co-effect between DON and AFB1 on HepG2/C3A cells were classified as being additive. Flow cytometry assay on the rate of apoptosis also shown an additive interaction between the two toxins at 0.56 μmol/Land 2.5 μmol/L for DON and AFB1 which could indicated that these two toxins could induced apoptosis and they also induced cycle arrest in S-phase or S, G2/M- phase. The increase of expression of cell apoptosis related genes of caspase-3 and Bax as well as the decrease of Bcl-2 further support their additive toxic effect on HepG2/C3A cells.
Keywords: Deoxynivalenol Alfatoxin B1 apoptosis combined toxicity
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