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目的 探讨低剂量无机砷对人正常肝细胞(L-02)中Sirt1及细胞周期相关基因表达的影响.方法 0.0(对照)、0.1、0.2、0.4 μmol/L三氧化二砷(As2O3)处理L-02细胞24、48、72 h,CCK-8法检测砷剂对细胞增殖的影响;蛋白质免疫印迹法(Western blot)检测砷对Sirt1蛋白表达及细胞内分布的影响;实时荧光定量PCR(qRT-PCR)检测砷对Sirt1及细胞周期相关基因p16、p21和p53 mRNA表达的影响;染色质免疫共沉淀(ChIP)技术检测砷对细胞周期相关基因启动子区H3K9、H3K27乙酰化及Sirt1富集程度的影响.结果 ①不同剂量砷分别作用于L-02细胞24、48、72 h,均可促进细胞的增殖(P均＜0.05),增幅在15％～40％.②染砷48 h,0.1 μmol/L砷处理组p16、p21、p53 mRNA表达(0.80±0.10、0.88±0.02、0.85±0.05)低于对照组(1.00±0.04、1.00±0.03、1.00±0.06,t=-3.22、-5.76、-3.23,P均＜0.05);p16、p21、p53基因启动子区H3K9、H3K27乙酰化富集程度均低于对照组(t=-2.89、-9.50、-17.64、-2.88、-3.41、-3.73,P均＜0.05),降低幅度在20％～ 70％.③染砷48 h,0.1 μmol/L砷处理组Sift1在细胞核内分布(1.04±0.02)高于对照组(1.00±0.01,t=3.10,P＜ 0.05),平均增加4％;p16、p53基因启动子区Sirt1的富集程度(1.33±0.04、4.28±0.12)高于对照组(1.00±0.13、1.00±0.13,t=4.20、32.11,P均＜0.05),至少增加30％.结论 低剂量无机砷可能通过促进Sirt1的核内分布,降低p16、p21、p53细胞周期相关基因启动子区H3K9、H3K27乙酰化的富集水平,抑制p16、p21、p53细胞周期相关基因的表达,从而促进细胞的增殖.“,”Objective To investigate the effect of low dose arsenic on expression of Sirt1 and cell cycle related genes in human normal hepatocytes (L-02).Methods After L-02 cells were treated with 0.0 (control),0.1,0.2 and 0.4 μmol/L As2O3 for 24,48 and 72 h,the effects of arsenic on cell proliferation were detected by CCK-8 assay,the effects of arsenic on Sirt1 protein expression and the intracellular distribution of Sirt1 protein were detected by Western blot,and the effects of arsenic on the expression of Sirt1,p16,p21 and p53 mRNA were detected by quantitative real-time PCR (qRT-PCR).In parallel,the effects of arsenic on acetylation of H3K9,H3K27 and the enrichment degree of Sirt1 in the promoter region of the cell cycle related genes were detected by chromatin immunoprecipitation (CHIP).Results (①)Treatment of different doses of arsenic on L-02 cells for 24,48 and 72 h promoted the proliferation of cells up to 15％ to 40％ (all P ＜ 0.05).(②)After arsenic exposure for 48 h,compared with the control group (1.00 ± 0.04,1.00 ± 0.03,1.00 ± 0.06),the expression of p16,p21 and p53 mRNA were down regulated in 0.1 μmol/L arsenic treated group (0.80 ± 0.10,0.88 ± 0.02,0.85 ± 0.05,t =-3.22,-5.76,-3.23,all P ＜ 0.05);compared with the control group,acetylation of H3K9 and H3K27 of p16,p21 and p53 genes in 0.1 μmol/L arsenic treated group were down regulated (t =-2.89,-9.50,-17.64,-2.88,-3.41,-3.73,all P ＜ 0.05),and the reduction amplitude was about 20％ to 70％.(③)After arsenic exposure for 48 h,compared with the control group (1.00 ± 0.01),the intracellular distribution of Sirt1 protein was up regulated in 0.1 μmoFL arsenic treated group (1.04 ± 0.02,t =3.10,P ＜ 0.05),with an average increase of 4％;the enrichment degree of Sift1 in the promoter region of p16 and p53 of 0.1 μmol/L arsenic treated group (1.33 ± 0.04,4.28 ± 0.12) was significantly higher than those in the control group (1.00 ± 0.13,1.00 ± 0.13,t =4.20,32.11,all P ＜ 0.05),with an increase of at least 30％.Conclusion Low-dose of inorganic arsenic could inhibit the expression of cell cycle related genes p16,p21 and p53 and reduce the accumulation of H3K9 and H3K27 acetylation by promoting the Sirt1 nuclear distribution,thereby promoting cell proliferation.