低氧预处理及AMPK过表达对过氧化氢抑制牙髓细胞增殖影响的研究

Effects of hypoxia preconditioning and AMPK overexpression on proliferative inhibition of dental pulp cells by hydrogen perioxide

目的:观察低氧预处理以及腺苷酸单磷酸激活蛋白激酶(AMPK)过表达对过氧化氢(H2O2)抑制牙髓细胞增殖的影响。方法:牙髓细胞分别在常氧(950 mL/L O2)和低氧(10 mL/L O2)环境下培养12 h后,用qRT-PCR检测细胞中AMPK mRNA的表达水平;分别取常氧和低氧培养12 h的牙髓细胞与0.1 mmol/L H2O2共培养4 h后,常规条件下继续培养4 d,用MTT法检测细胞的增殖能力;分别用AMPK过表达质粒和特异性小RNA干扰(siRNA)片段转染牙髓细胞,与0.1 mmol/L H2O2共培养4 h,然后常规条件下继续培养4 d,用MTT法检测细胞的增殖能力。结果:低氧预处理能显著上调牙髓细胞中AMPK mRNA的表达水平,并能减轻H2O2对细胞增殖的抑制作用(P<0.05);AMPK过表达细胞经过H2O2处理后,增殖能力明显高于正常细胞+H2O2组和siRNA+H2O2组(P<0.05),而siRNA干扰的细胞经H2O2处理后其增殖能力则较其他各组降低(P<0.05)。结论:低氧预处理和AMPK基因的上调可使牙髓细胞获得对H2O2氧化的抗性,从而减轻H2O2对细胞增殖的抑制作用。

AIM：To investigate the effects of hypoxia preconditioning and AMP-activated protein kinase （AMPK）overexpression on proliferative inhibition of dental pulp cells by hydrogen perioxide（H2O2）.METHODS：The expression of AMPK mRNA in dental pulp cells was analyzed by qRT-PCR after the cells had been exposed to nor-moxia （950 mL/L O2 ）or hypoxia （10 mL/L O2 ）for 12 h.The cells were then cocultured with 0.1 mmol/L H2 O2 for 4 h and cell proliferation was evaluated by MTT assay.Furthermore,the cells were transfected with AMPK overexpres-sion vector and specific siRNA respectively.Then transfected cells were cocultured with 0.1 mmol/L H2 O2 for 4 h,the proliferation of the cells was evaluated by MTT assay.RESULTS：Hypoxia preconditioning upregulated AMPK mRNA levels in dental pulp cells and ameliorated proliferative inhibition by H2 O2 （P〈0.05 ）.The cells transfected with AMPK overexpression vector attained higher proliferative rate than the controls or those transfected with siRNA after challenging with H2 O2 .However,AMPK knockdown significantly inhibited proliferation of the cells （P 〈0.05 ）. CONCLUSION：Hypoxia preconditioning and AMPK up-regulation may confer resistance to oxidation,hence de-crease the proliferation inhibition induced by H2 O2 in dental pulp cells.