摘要:
为了研究新型持久性有机污染物全氟辛烷磺酸(perffluorooctane sulfonate,PFOS)的神经毒性及其机制,将PC12细胞暴露于31.25、62.5、125、250、500μmol·L-1的PFOS,利用噻唑蓝(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT)法检测细胞存活率、二氯二氢荧光素-乙酰乙酸酯(2',7'-Dichlorofluorescin diacetate,DCFH-DA)荧光探针检测活性氧含量、Hoechst 33342/PI试剂盒检测细胞凋亡及坏死情况、溶酶体特异性荧光染料Lyso-Tracker Red监测细胞内溶酶体数量;利用实时荧光定量PCR(quantitative real-time reverse transcription-polymerase chain reaction,q RT-PCR)技术对自噬发生的代表性基因Atg5、Atg 12、Beclin 1的表达情况进行考察。结果发现,与对照组比较,250、500μmol·L-1PFOS显著降低PC12细胞存活率(P<0.05);62.5、125、250、500μmol·L-1PFOS诱导活性氧含量升高(P<0.05);62.5、125、250、500μmol·L-1PFOS诱导PC12细胞凋亡(P<0.05);125、250、500μmol·L-1PFOS升高溶酶体数量及Atg 5、Atg 12、Beclin 1基因表达(P<0.05)。结果表明,PFOS能够诱导PC12细胞死亡,其机制可能为干扰自噬与凋亡过程。
关键词:
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PFOS
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PC12
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神经毒性
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凋亡
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自噬
Abstract:
To investigate the neurotoxic effect ofperfluorooctane sulfonate 00FOS)and its underlying mechanism, the PC12 cells were exposed to 3125, 62.5,125,250 and 500 μmol·L-1 PFOS. Cells were then evaluated on viability using MTT, generation of reactive oxygen species (ROS) using DCFH-DA, apoptosis by Hoechst 33342/PI kit, and number of lysosomes by specific dye, Lyso-Tracker Red. We further employed the real time RT-PCR to evaluate some genes that may differentially express during autophagy, such as Atg 5, Atg 12 and Beclin 1. Compared to control cells, the viability of ceils exposed to 250 or 500 μmol·L-1 PFOS was significantly decreased (P<0.05), and the increases in apoptosis and ROS production were observed in cells exposed to 62.5 μmol·L-1 PFOS and above. There were also increases in the number of lysosomes and the gene expres-sion of Atg 5, Atg 12 and Beclin 1 in cells exposed to 125,250,500 μmol·L-1 PFOS (P<0.05), respectively. Our data suggest that autophagy and apoptosis might be involved in PFOS-induced death in PC12.
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