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目的检测尿嘧啶DNA糖苷酶2(UNG2)的糖苷酶活性,研究UNG2在肝癌细胞HepG2抗氧化损伤过程中的作用。方法构建UNG2的过表达载体,利用Western blot法检测UNG2过表达效果。在HepG2细胞过表达UNG2,免疫荧光细胞化学染色观察UNG2在细胞中的表达和定位,利用含脱氧尿苷的寡核苷酸为底物测定UNG2的DNA糖苷酶活性,利用H_2O_2毒性实验研究UNG2在HepG2肝癌细胞抗氧化存活中的作用。结果在HEK293FT细胞中成功表达了UNG2,并发现UNG2主要定位在HepG2细胞核中。酶活性实验表明UNG2可以有效地切割寡核苷酸中的脱氧尿苷位点。H_2O_2毒性实验表明过表达UNG2可以显著提高HepG2肝癌细胞在H_2O_2处理后的存活率。结论 UNG2具有特异的尿嘧啶糖苷酶活性,并且UNG2能够保护HepG2肝癌细胞抵抗氧化应激损伤。
Objective To detect the glycosidase activity of uracil DNA glycosylase 2 (UNG2) and study the role of UNG2 in the anti-oxidative damage of HepG2 cells. Methods The UNG2 overexpression vector was constructed and the effect of UNG2 overexpression was detected by Western blot. UNG2 was overexpressed in HepG2 cells, the expression and localization of UNG2 in cells were observed by immunofluorescence cytochemistry, the DNA glycosidase activity of UNG2 was determined by using deoxyuridine-containing oligonucleotides as substrate, and the effect of UNG2 on Antioxidative survival of HepG2 hepatoma cells. Results UNG2 was successfully expressed in HEK293FT cells and found that UNG2 was predominantly located in the nucleus of HepG2 cells. Enzyme activity experiments show that UNG2 can effectively cleave the deoxyuridine site in the oligonucleotide. H_2O_2 toxicity experiments showed that over-expression of UNG2 could significantly improve the survival rate of HepG2 hepatoma cells treated with H_2O_2. Conclusion UNG2 has specific uracil glycosidase activity, and UNG2 can protect HepG2 liver cancer cells against oxidative stress injury.