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目的探讨镉对蛋白磷酸酶2A(PP2A)的B55δ亚基高表达和不同多态性单体型调控高表达的肝细胞周期的影响及其与核因子-kappaB(NF-κB)活化的关系。方法选择永生化人正常肝L02细胞,采用B55δ编码基因PPP2R2D序列构建高表达的L02-2R2Dc细胞、与不同启动子区单体型调控高表达的L02-2R2D-PC1和L02-2R2D-PC3细胞株,以导入空载体的L02-pBabe为对照细胞。各细胞给予5～80μmol/L的氯化镉(CdCl2)处理建立细胞模型。噻唑蓝法检测各细胞生长的抑制作用;流式细胞术检测细胞周期;蛋白免疫印迹(Western blotting)检测细胞核内NF-κBp65蛋白的活化情况;荧光定量聚合酶链反应(QRT-PCR)检测环氧合酶-2(COX-2)基因mRNA水平的改变。结果与对照组比较,CdCl2组各细胞的增殖抑制率随镉水平增高呈剂量依赖性增高(Pearson’s r均P<0.05);与CdCl2组比较,冈田酸+CdCl2组各细胞的增殖抑制率明显增加(P<0.05);与对照组比较,CdCl2组细胞G1期比例下降、S期比例增加(P<0.05);胞核p65蛋白水平增高,COX-2 mRNA水平上调;这种效应在不同细胞株之间比较,差异有统计学意义(P<0.05)。结论镉可引起PP2A-B55δ高表达的人肝L02细胞增殖功能抑制并导致细胞周期S期阻滞,这种效应与NF-κB信号通路的活化有关。
Objective To investigate the effect of cadmium on the expression of B55δ subunit of protein phosphatase 2A (PP2A) and the cell cycle regulated by haplotypes with different polymorphisms and its relationship with the activation of nuclear factor-kappaB (NF-κB). Methods Human immortalized human L02 cells were immortalized. The L02-2R2D2 cells were constructed by using the B55δ-encoding gene PPP2R2D sequence. L02-2R2Dc cells with high expression level under different haplotypes of promoter region were identified as L02-2R2D-PC1 and L02-2R2D-PC3 L02-pBabe, which was introduced into empty vector, served as control cells. The cells were treated with 5 ~ 80μmol / L cadmium chloride (CdCl2) to establish a cell model. The inhibition of cell growth was detected by thiazolyl blue staining. The cell cycle was detected by flow cytometry. The activation of NF-κBp65 in nuclei was detected by Western blotting. The expression of NF-κBp65 in nuclei was detected by fluorescence quantitative polymerase chain reaction (QRT-PCR) Oxygenase-2 (COX-2) gene mRNA level changes. Results Compared with the control group, the proliferation inhibition rate of each cell in CdCl2 group increased in a dose-dependent manner with increasing of cadmium concentration (Pearson’s r all P <0.05). Compared with CdCl2 group, the inhibition rate of okadaic acid + CdCl2 group was significantly (P <0.05). Compared with the control group, the percentage of G1 phase in CdCl2 group decreased and the proportion of S phase increased (P <0.05), the level of nuclear p65 protein increased and the level of COX-2 mRNA increased in CdCl2 group The difference between the strains was statistically significant (P <0.05). Conclusion Cadmium can inhibit the proliferation of L02 cells with high expression of PP2A-B55δ and result in cell cycle arrest. This effect is related to the activation of NF-κB signaling pathway.