發(fā)布時(shí)間：2018-04-22 23:03

  本文選題：二氫生物蝶呤還原酶(QDPR) + 轉(zhuǎn)化生長(zhǎng)因子-β(TGF-β)��； 參考：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》2017年09期

【摘要】：目的:評(píng)估二氫生物蝶呤還原酶(QDPR)的抗氧化作用,并初步探討QDPR基因A278C位點(diǎn)突變對(duì)其抗氧化作用的影響。創(chuàng)新點(diǎn):首次在體外實(shí)驗(yàn)中發(fā)現(xiàn)QDPR有抗氧化作用,且此作用在A278C位點(diǎn)突變后減弱。方法:我們構(gòu)建了野生型和突變型QDPR質(zhì)粒,且分別轉(zhuǎn)染至人胚腎293細(xì)胞中(HEK293T)。實(shí)驗(yàn)可分為以下三組:空白質(zhì)粒對(duì)照組、野生型QDPR組和突變型QDPR組。三天后收集細(xì)胞觀察活性氧(ROS)和四氫生物蝶呤(BH4)的表達(dá)量,使用免疫印跡的方法檢測(cè)煙酰胺腺嘌呤二核苷酸磷酸氧化酶4(NOX4)、谷胱甘肽過(guò)氧化物酶3(GPX3)和超氧化物歧化酶1(SOD1)的蛋白表達(dá)水平。用半定量逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)方法分析神經(jīng)型一氧化氮合成酶(n NOS)基因的表達(dá)。用酶聯(lián)免疫吸附測(cè)定(ELISA)試劑盒檢測(cè)轉(zhuǎn)化生長(zhǎng)因子-β1(TGF-β1)的活性。結(jié)論:本實(shí)驗(yàn)中野生型QDPR可以顯著降低n NOS、NOX4和TGF-β1的水平,同時(shí)提高SOD1和GPX3表達(dá)。但當(dāng)QDPR發(fā)生位點(diǎn)突變后沒(méi)有觀察到上述現(xiàn)象,并且突變型會(huì)導(dǎo)致ROS過(guò)量產(chǎn)生。我們的數(shù)據(jù)還表明,野生型和突變型QDPR對(duì)BH4含量的影響無(wú)顯著差異。綜上所述,QDPR有抗氧化作用,但A278C位點(diǎn)突變后會(huì)影響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.
【作者單位】： 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)
【分類號(hào)】：R587.2
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本文編號(hào)：1789323