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为探讨氧化应激对人骨肉瘤细胞增殖的影响及其作用机理,首先用H2O2处理U2OS细胞,采用Western印迹和real-time PCR检测HMG盒转录因子1(HBP1)及其下游靶基因DNMT1和p16表达水平的变化.用荧光素酶报告基因实验检测在H2O2诱导下,HBP1对于DNMT1和p16启动子的影响.用细胞增殖试验(BrdU掺入,细胞生长曲线)检测H2O2对细胞增殖的影响以及HBP1的作用.用衰老相关β半乳糖苷酶(SA-β-Gal)染色检测在H2O2诱导的细胞衰老中HBP1所起的作用.Western印迹,real-time PCR及荧光素酶报告基因实验结果显示,细胞经H2O2处理后,明显增高HBP1表达水平,转录抑制DNMT1的表达,促进p16蛋白的表达.细胞增殖实验结果显示,H2O2显著抑制了细胞的增殖,HBP1 knockdown可部分逆转这种抑制作用.SA-β-Gal染色实验说明,H2O2可诱导HBP1表达正常的U2OS细胞衰老,而HBP1 knockdown使这种促衰老作用减弱.研究结果说明,H2O2可抑制人骨肉瘤细胞增殖,诱导细胞衰老.其作用机制是通过上调转录因子HBP1的表达,转录抑制或促进其下游靶基因DNMT1或p16的表达来抑制细胞增殖,促进细胞衰老.
To investigate the effect of oxidative stress on human osteosarcoma cell proliferation and its mechanism, U2OS cells were treated with H2O2 at first. The expression of HMG box transcription factor 1 (HBP1) and its downstream target genes DNMT1 and p16 were detected by Western blot and real-time PCR The effect of H2P1 on DNMT1 and p16 promoters was detected by luciferase reporter assay.Cell proliferation assay (BrdU incorporation and cell growth curve) was used to detect the effect of H2O2 on cell proliferation and HBP1 The role of HBP1 in H2O2-induced cell senescence was tested by aging-associated β-galactosidase (SA-β-Gal) staining.Western blotting, real-time PCR and luciferase reporter assay showed that cells H2O2 treatment significantly increased the expression of HBP1, and inhibited the expression of DNMT1 by transcription, which promoted the expression of p16 protein.Experimental results of cell proliferation showed that H2O2 significantly inhibited cell proliferation and HBP1 knockdown partially reversed the inhibition -Gal staining showed that H2O2 could induce the senescence of U2OS cells with normal expression of HBP1, and HBP1 knockdown reduced this pro-aging effect.The results showed that H2O2 could inhibit the proliferation of human osteosarcoma cells Proliferation and induce cell senescence.The mechanism of action is to inhibit cell proliferation and promote cell senescence by up-regulating the expression of HBP1, inhibiting transcription or promoting the expression of its downstream target gene DNMT1 or p16.