摘要
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 investigate the uracil glycosidic enzyme activity of uracil DNA glycosylase 2 （UNG2） and study the role of UNG2 in the resistance of antioxidant stress of HepG2 cells. Methods The UNG2-expressing vector was built. Western blotting was used to detect the expression of UNG2. Immunofluorescence staining was performed to observe the cellular location of UNG2. Oligonucleotide was used as substrate for the determination of the UNG2 glycosidic enzyme activity. H202 toxicity assay was done to study the function of UNG2 in the antioxidant resistance of hepatocellular carcinoma HepG2 cells. Results UNG2 was successfully over-expressed in HEK293FT cells, and UNG2 was found to be mainly located in nucleus. Enzyme activity assay showed that UNG2 had significant oligonucleotide dU glycosidic enzyme activity. H202toxicity assay showed that over-expressed UNG2 could remarkably increase the survival of HepG2 cells after exposed to H202. Conclusion UNG2 possesses specific DNA glycosidic enzyme activity, and it can protect HepG2 cells against oxidative stress damage.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2017年第4期483-487,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(31570168)
关键词
UNG2
DNA糖苷酶
酶活性
氧化应激
uracil DNA glycosylase 2 （UNG2）
DNA glycosidase
enzyme activity
oxidative stress
