摘要
目的研究高糖状态下核转录因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)对人甲状腺乳头状癌K1细胞凋亡的影响,并探讨氧化应激在Nrf2影响K1细胞凋亡中的作用。方法 25 mmol/L高糖条件下,将甲状腺乳头状癌K1细胞分为对照组、Nrf2siRNA组(转染siRNA抑制Nrf2的表达)、NcsiRNA组(阴性转染),培养48 h后,流式细胞仪检测细胞凋亡率,western blot检测Nrf2(核和浆蛋白)及凋亡相关蛋白B淋巴细胞瘤基因2(B-cell lymphoma-2,Bcl-2)和半胱氨酸蛋白酶3(Caspase-3)表达水平,逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测Nrf2mRNA表达水平,相关试剂盒检测活性氧(reactive oxygen species,ROS)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)水平。结果与对照组比较,Nrf2siRNA组K1细胞早期凋亡率和晚期凋亡率明显增加(P均<0.01),K1细胞Nrf2mRNA、核蛋白及浆蛋白表达水平明显下降(P均<0.01),K1细胞抗凋亡相关蛋白Bcl-2水平下降、促凋亡相关蛋白Caspase-3水平增加(P均<0.01),K1细胞ROS水平增加、GSH及SOD水平下降(P均<0.01);NcsiRNA组上述指标与对照组比较均无统计学差异(P均>0.05)。结论高糖状态下,沉默Nrf2的表达可以增强K1细胞氧化应激水平、下调抗凋亡相关蛋白的表达、上调促凋亡相关蛋白的表达,促进K1细胞凋亡,提示Nrf2可能通过抗氧化应激损伤抑制高糖状态下K1细胞的凋亡。
Objective To study the effect of nuclear factor erythroid 2-related factor 2(Nrf2) on the apoptosis of human papillary thyroid carcinoma K1 cells under high glucose environmental conditions, and investigate the role of oxidative stress in the apoptosis of K1 cells regulated by Nrf2. Methods K1 cells were cultured with 25 mmol/L glucose and then divided into three groups:the control group, Nrf2 siRNA group, and NcsiRNA group.After 48 hours of culturing,the apoptosis rate was detected by flow cytometry.The expression levels of Nrf2(nuclear and cytoplasmic protein),the apoptosis-related proteins B-cell lymphoma-2(Bcl-2) and Caspase-3 were detected by western blot. The expression of Nrf2 mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR).The levels of reactive oxygen species(ROS),superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) were detected by related kits. Results Compared with control group, the early apoptosis and late apoptosis of K1 cells in Nrf2 siRNA group were significantly increased(all P<0.01),the expression levels of Nrf2 mRNA,nuclear protein and cytoplasmic protein of K1 cells in Nrf2 siRNA group were significantly decreased(all P<0.01),the protein level of Bcl-2 was decreased and Caspase-3 was increased in Nrf2 siRNA group(all P<0.01), the level of ROS was increased and the levels of GSH-Px and SOD were decreased in Nrf2 siRNA group(all P<0.01).All above test indicators had no significant difference between control group and NcsiRNA group(all P>0.05). Conclusion In high glucose state,silencing Nrf2 can enhance K1 cells oxidative stress, down-regulate the expression of anti-apoptosis related proteins and up-regulate the expression of pro-apoptosis related proteins,and can promote the apoptosis of K1 cells.It suggests that Nrf2 may inhibit the apoptosis of K1 cells in the high glucose state through antioxidant stress damage.
引文
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