A278C位点突变减弱了二氢生物蝶呤还原酶的抗氧化作用（英文）

发布时间：2018-04-22 23:03

  本文选题：二氢生物蝶呤还原酶(QDPR) + 转化生长因子-β(TGF-β)　； 参考：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》2017年09期

【摘要】：目的:评估二氢生物蝶呤还原酶(QDPR)的抗氧化作用,并初步探讨QDPR基因A278C位点突变对其抗氧化作用的影响。创新点:首次在体外实验中发现QDPR有抗氧化作用,且此作用在A278C位点突变后减弱。方法:我们构建了野生型和突变型QDPR质粒,且分别转染至人胚肾293细胞中(HEK293T)。实验可分为以下三组:空白质粒对照组、野生型QDPR组和突变型QDPR组。三天后收集细胞观察活性氧(ROS)和四氢生物蝶呤(BH4)的表达量,使用免疫印迹的方法检测烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)、谷胱甘肽过氧化物酶3(GPX3)和超氧化物歧化酶1(SOD1)的蛋白表达水平。用半定量逆转录-聚合酶链反应(RT-PCR)方法分析神经型一氧化氮合成酶(n NOS)基因的表达。用酶联免疫吸附测定(ELISA)试剂盒检测转化生长因子-β1(TGF-β1)的活性。结论:本实验中野生型QDPR可以显著降低n NOS、NOX4和TGF-β1的水平,同时提高SOD1和GPX3表达。但当QDPR发生位点突变后没有观察到上述现象,并且突变型会导致ROS过量产生。我们的数据还表明,野生型和突变型QDPR对BH4含量的影响无显著差异。综上所述,QDPR有抗氧化作用,但A278C位点突变后会影响QDPR的抗氧化功能。
[Abstract]:Aim: to evaluate the antioxidation effect of dihydrobiopterin reductase (QDPR) and to explore the effect of A278C mutation of QDPR gene on its antioxidation. Innovation: for the first time, QDPR was found to have antioxidant effect in vitro, and this effect was weakened after mutation at A278C. Methods: wild-type and mutant QDPR plasmids were constructed and transfected into human embryonic kidney 293 cells respectively. The experiment was divided into three groups: blank plasmid control group, wild type QDPR group and mutant QDPR group. After three days, cells were collected to observe the expression of Ros) and tetrahydrobiopterin (BH4). The protein expression levels of nicotinamide adenine dinucleotide phosphate oxidase (4NOX4), glutathione peroxidase 3 (GPX3) and superoxide dismutase 1 (SOD1) were detected by Western blot. The expression of neuronal nitric oxide synthase (nNOS) gene was analyzed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Enzyme linked immunosorbent assay (Elisa) was used to detect the activity of transforming growth factor-尾 1 (TGF- 尾 1). Conclusion: in this experiment, wild type QDPR can significantly reduce the levels of nNOSn, NOX4 and TGF- 尾 1, and increase the expression of SOD1 and GPX3 at the same time. However, the above phenomenon was not observed after the mutation of QDPR site, and the mutation type resulted in excessive production of ROS. Our data also showed that there was no significant difference between wild type QDPR and mutant QDPR on BH4 content. In conclusion, QDPR has antioxidant effect, but A278C mutation will affect the antioxidant function of QDPR.
【作者单位】： Beijing
【基金】：supported by the National Natural Science Foundation of China(No.81130066) the International Cooperation and Exchanges of the National Natural Science Foundation of China(No.81620108031)
【分类号】：R587.2
，

本文编号：1789323