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目的 :研究二烯丙基三硫(diallyl trisuli de,DATS)对人白血病细胞HL-60细胞中还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate,NADPH)氧化酶活性的影响及可能的作用机制。方法:DATS(150μmol/L)作用于HL-60细胞不同时间后,采用硝基四氮唑蓝(nitro blue-tetrazolium,NBT)还原实验检测NADPH氧化酶的活性;实时荧光定量PCR检测DATS对微RNA-34a(microRNA-34a,miR-34a)以及Src、Gab1和Shp-2 mRNA表达的影响及miR-34a对Src、Gab1和Shp-2 mRNA表达的影响;蛋白质印迹法检测DATS及miR-34a对Src、Gab1和Shp-2蛋白磷酸化的影响;细胞计数实验检测DATS和miR-34a对HL-60细胞增殖的影响。结果 :DATS作用于HL-60细胞后能明显提高细胞中NADPH氧化酶的活性,且呈时间依赖性,而Src激酶抑制剂PP2可抑制这种作用;DATS能上调HL-60细胞中miR-34a的表达水平,呈时间依赖性;miR-34a过表达可抑制Src、Gab1和Shp-2 mRNA及磷酸化蛋白的表达;DATS能抑制磷酸化Src、Gab1和Shp-2蛋白的表达水平。DATS和miR-34a过表达能抑制HL-60细胞的增殖,而抑制miR-34a的表达则可提高白血病细胞的增殖能力。结论 :DATS可能通过促进HL-60细胞miR-34a-Src-Gab1-Shp途径调控NADPH的氧化酶活性,从而起到抑制白血病细胞增殖的作用。
Objective: To investigate the effect of diallyl trisuli de (DATS) on the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in human leukemia HL-60 cells. Possible mechanism of action. Methods: After treated with DATS (150μmol / L) for different time, the activity of NADPH oxidase was detected by nitro blue-tetrazolium (NBT) reduction assay. The real-time quantitative PCR The effect of miR-34a on the expression of Src, Gabl and Shp-2 mRNA was examined by Western blotting. The expressions of miR-34a, miR-34a, miR-34a and miR- On the phosphorylation of Src, Gab1 and Shp-2, and the cell counting experiments were used to detect the effect of DATS and miR-34a on the proliferation of HL-60 cells. Results: DATS could significantly increase the activity of NADPH oxidase in HL-60 cells in a time-dependent manner, while Src kinase inhibitor PP2 could inhibit this effect. DATS could up-regulate the expression of miR-34a MiR-34a overexpression inhibited the expression of Src, Gab1 and Shp-2 mRNA and phosphorylated protein; DATS inhibited the phosphorylation of Src, Gab1 and Shp-2 protein expression. Overexpression of DATS and miR-34a can inhibit the proliferation of HL-60 cells, while the inhibition of miR-34a expression can increase the proliferation of leukemia cells. CONCLUSION: DATS may inhibit the proliferation of leukemic cells by regulating the oxidase activity of NADPH through the miR-34a-Src-Gab1-Shp pathway in HL-60 cells.