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目的研究水通道蛋白9(AQP9)mRNA表达水平、水通道蛋白9及p38蛋白表达及其磷酸化水平对肝癌细胞HepG2和肝正常细胞L-02砷摄入的影响,探讨水通道蛋白9磷酸化的调控机制。方法采用电感藕合等离子体质谱法(ICP-MS)测定细胞内砷含量。采用实时定量PCR、免疫印迹法和免疫共沉淀技术分别检测不同处理后两细胞株中水通道蛋白9 mRNA、水通道蛋白9和p38蛋白表达水平及其磷酸化水平。采用SPSS统计软件分析实验数据。结果 HepG2细胞内砷含量及摄入速度高于L-02细胞。HepG2细胞的水通道蛋白9 mRNA表达水平在6 h内随NaAsO2处理时间延长而显著增加(P<0.05),而在L-02细胞无明显变化。在2 h时,HepG2细胞水通道蛋白9基因表达水平在各NaAsO2处理浓度均显著增加(P<0.05)。免疫沉淀实验结果显示,HepG2细胞中水通道蛋白9蛋白磷酸化水平随NaAsO2处理时间和浓度的增加而增加,而L-02细胞在各时间和浓度处理点水通道蛋白9蛋白磷酸化水平与对照相比明显增加,但处理间无明显差异;p38的磷酸化水平在两种细胞中均随砷处理时间延长而增加;SB203580抑制p38活性后能完全取消L-02细胞水通道蛋白9蛋白的磷酸化,而对HepG2细胞水通道蛋白9蛋白磷酸化无明显影响。结论水通道蛋白9的表达及磷酸化水平可能在调节细胞砷摄入中发挥重要作用,在不同细胞中水通道蛋白9蛋白磷酸化的调控机制有所差异。
Objective To investigate the effects of aquaporin 9 (AQP9) mRNA expression, aquaporin 9 and p38 protein expression and phosphorylation on the uptake of arsenic in HepG2 and L-02 cells, and to investigate the effects of aquaporin 9 phosphorylation The regulatory mechanism. Methods Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the intracellular arsenic content. Real-time quantitative PCR, Western blotting and co-immunoprecipitation were used to detect the expression and phosphorylation of aquaporin 9 mRNA, aquaporin 9 and p38 protein in the two cell lines respectively. SPSS statistical software analysis of experimental data. Results The arsenic content and uptake rate of HepG2 cells were higher than that of L-02 cells. The expression level of aquaporin 9 mRNA in HepG2 cells increased significantly with the prolongation of NaAsO2 treatment (P <0.05) within 6 h, but not in L-02 cells. At 2 h, the expression level of aquaporin 9 in HepG2 cells was significantly increased at each concentration of NaAsO2 (P <0.05). The results of immunoprecipitation showed that the phosphorylation of aquaporin 9 in HepG2 cells increased with the time and concentration of NaAsO2 treatment, whereas the phosphorylation of aquaporin 9 in L-02 cells at each time and concentration But there was no significant difference between treatments. The phosphorylation level of p38 increased with the time of arsenic treatment in both cells. SB203580 could completely abolish the phosphorylation of aquaporin 9 in L-02 cells after inhibiting the activity of p38 While no significant effect on aquaporin 9 phosphorylation in HepG2 cells. Conclusion The expression and phosphorylation of aquaporin 9 may play an important role in regulating cellular arsenic uptake. The regulatory mechanisms of aquaporin 9 phosphorylation in different cells are different.