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肝癌细胞蛋白磷酸酶2A催化亚基在过氧化氢和黄曲霉毒素B1诱导急慢性肝癌细胞损伤模型中的表达及其对肝癌细胞的影响
DOI: 10.3969/j.issn.1001-5256.2019.12.018
Expression of protein phosphatase 2A catalytic subunit in hepatoma cells after acute and chronic injury induced by hydrogen peroxide and aflatoxin B1 and its influence on hepatoma cells
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摘要: 目的探讨过氧化氢和黄曲霉毒素B1诱导肝癌细胞急慢性损伤后蛋白磷酸酶2A催化亚基（PP2A）的表达情况。方法采用不同浓度的过氧化氢（0、50、100、200、400、800μmol/L）或黄曲霉毒素B1（0、1. 25、2. 5、5、10、20、40μmol/L）诱导肝癌细胞（HepG2/Huh-7细胞） 2、4、6、8和24、48 h后,倒置显微镜观察细胞形态,刃天青实验检测肝癌细胞活性,Western Blot检测去甲基化PP2A催化亚基C（PP2Ac）和总PP2Ac蛋白的表达情况。两组间比较用独立样本t检验,多组间比较采用单因素方差分析和析因设计资料两因素方差分析。结果 HepG2/Huh-7细胞对过氧化氢和黄曲霉毒素B1诱导敏感,随着过氧化氢和AFB1浓度升高或孵育时间延长,细胞形态发生变化,数量减少。在2、4、6和8 h时间段,100μmol/L及以上浓度的过氧化氢诱导HepG2/Huh-7细胞后,随着过氧化氢浓度的升高,HepG2/Huh-7细胞活性逐渐下降,不同浓度过氧化氢与对照组（0μmol/L）比较,差异均有统计学意义（P值均<0. 05）。在4个不同时间段,两株...Abstract: Objective To investigate the expression of protein phosphatase 2 A catalytic subunit in hepatoma cells after acute and chronic injury induced by hydrogen peroxide and aflatoxin B1. Methods Hepatoma cells( HepG2/Huh-7 cells) were treated with different concentrations of hydrogen peroxide( 0,50,100,200,400,and 800 μmol/L) for 2,4,6,and 8 hours or aflatoxin B1( 0,1. 25,2. 5,5,10,20,and 40 μmol/L) for 24 and 48 hours. An inverted microscope was used to observe cell morphology,resazurin assay was used to measure the activity of hepatoma cells,and Western blot was used to measure the expression of demethylated PP2 Ac( dem-PP2 Ac) and total PP2 Ac protein. The independent samples t-test was used for comparison between two groups,a one-way analysis of variance was used for comparison between multiple groups,and the two-way analysis of variance was used for factorial design data. Results HepG2/Huh-7 cells were sensitive to hydrogen peroxide and aflatoxin B1,and with the increases in the concentration of hydrogen peroxide and AFB1 concentration or the prolonged incubation time,there were changes in cell morphology and a reduction in the number of cells. At 2,4,6,and 8 hours of the treatment with hydrogen peroxide at a concentration of 100 μmol/L or above,the activity of HepG2/Huh-7 cells gradually decreased with the increase in the concentration of hydrogen peroxide,and the one-way analysis of variance showed a significant difference between suchcells and the control group( all P < 0. 05). The two-way analysis of variance showed no significant difference in IC50 between the two cell lines during these four periods of time( P > 0. 05). At 24 and 48 hours of the treatment with aflatoxin B1 at a concentration of 2. 5 μmol/L or above,the activity of HepG2/Huh-7 cells gradually decreased with the increase in the concentration of aflatoxin B1,and the one-way analysis of variance showed a significant difference between such cells and the control group( all P < 0. 05). The two-way analysis of variance showed no significant difference in IC50 between the two cell lines during these two periods of time( P > 0. 05). After HepG 2 cells were induced by 200 μmol/L hydrogen peroxide for 2 hours,this group of cells had significantly higher expression levels of total PP2 Ac protein and dem-PP2 Ac than the control group( t =-5. 553 and-2. 990,P = 0. 005 and 0. 040); after Huh-7 cells were induced by 400 μmol/L hydrogen peroxide for 2 hours,this group of cells had significantly higher expression levels of total PP2 Ac protein and dem-PP2 Ac than the control group( t = 0. 073 and-5. 149,P = 0. 002 and 0. 007). After HepG 2 cells were induced by 10 μmol/L aflatoxin B1 for 2 hours,this group of cells had significantly higher expression levels of total PP2 Ac protein and dem-PP2 Ac than the control group( t =-4. 561 and-5. 788,P = 0. 010 and 0. 004); after Huh-7 cells were induced by 20 μmol/L aflatoxin B1 for 2 hours,this group of cells had significantly higher expression levels of total PP2 Ac protein and dem-PP2 Ac than the control group( t =-5. 120 and-6. 144,P = 0. 007 and 0. 004).Conclusion Morphological changes,a reduction in cell activity,and increases in the expression of total PP2 Ac and dem-PP2 Ac are observed after acute and chronic injury of hepatoma cells induced by hydrogen peroxide and aflatoxin B1.
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Key words:
- carcinoma,hepatocellular /
- hydrogen peroxide /
- aflatoxin B1 /
- protein phosphatase 2 /
- HepG2 cells /
- Huh-7 cells
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