CAJ | 학술논문

目的探讨过表达NF-E2相关因子2(Nrf2)对黑素细胞线粒体合成和功能的影响.方法构建含有人Nrf2基因全长的过表达质粒Nrf2-pEX-1,用该质粒瞬时转染白癜风患者表皮黑素细胞系(PIG3V).实验分为空白组(不含质粒)、对照组(转染pEX-1空质粒)、过表达组(转染pEX-1-Nrf2过表达质粒).过表达Nrf2后,RT-PCR和Western印迹法检测PIG3V线粒体生物合成相关的Nrf2、核呼吸因子1(NRF1)、线粒体转录因子A(TFAM)mRNA和蛋白水平的变化;RT-PCR检测线粒体DNA(mtDNA)拷贝数.流式细胞仪检测线粒体膜电位(MMP);荧光素酶报告系统检测细胞ATP含量.采用两样本t检验进行统计学分析.结果 Nrf2过表达质粒转染PIG3V细胞后24 h,Nrf2 mRNA、NRF1 mRNA水平较对照组明显升高,差异有统计学意义(均P＜0.001),而TFAM mRNA水平与对照组比较差异无统计学意义;48 h后Nrf2 mRNA、TFAM mRNA水平较对照组升高,差异均有统计学意义(P＜0.05),而NRF1 mRNA水平与对照组差异无统计学意义.Western印迹法显示,转染后24 h,过表达组Nrf2、NRF1、TFAM蛋白表达与对照组比较差异均无统计学意义,48 h后过表达组Nrf2、NRF1、TFAM蛋白表达升高(P＜0.05).过表达组MMP在转染24 h后较对照组升高2.313％(t=5.546,P=0.005),48 h后升高14.872％(t=8.537,P=0.001).线粒体合成指标相对mtDNA拷贝数,在转染24 h后两组间差异无统计学意义(P＞0.05),48 h后过表达组明显高于对照组(t=5.760,P=0.005);细胞ATP含量在转染后24 h两组间差异无统计学意义(P＞0.05),48 h后过表达组ATP含量较对照组明显升高(t=22.040,P=0.008).结论过表达Nrf2可以促进黑素细胞线粒体生物合成相关基因和蛋白的表达,进而促进线粒体生物合成,上调线粒体功能相关的指标.
목적 탐토과표체NF-E2상관인자2(Nrf2)대흑소세포선립체합성화공능적영향.방법 구건함유인Nrf2기인전장적과표체질립Nrf2-pEX-1,용해질립순시전염백전풍환자표피흑소세포계(PIG3V).실험분위공백조(불함질립)、대조조(전염pEX-1공질립)、과표체조(전염pEX-1-Nrf2과표체질립).과표체Nrf2후,RT-PCR화Western인적법검측PIG3V선립체생물합성상관적Nrf2、핵호흡인자1(NRF1)、선립체전록인자A(TFAM)mRNA화단백수평적변화;RT-PCR검측선립체DNA(mtDNA)고패수.류식세포의검측선립체막전위(MMP);형광소매보고계통검측세포ATP함량.채용량양본t검험진행통계학분석.결과 Nrf2과표체질립전염PIG3V세포후24 h,Nrf2 mRNA、NRF1 mRNA수평교대조조명현승고,차이유통계학의의(균P＜0.001),이TFAM mRNA수평여대조조비교차이무통계학의의;48 h후Nrf2 mRNA、TFAM mRNA수평교대조조승고,차이균유통계학의의(P＜0.05),이NRF1 mRNA수평여대조조차이무통계학의의.Western인적법현시,전염후24 h,과표체조Nrf2、NRF1、TFAM단백표체여대조조비교차이균무통계학의의,48 h후과표체조Nrf2、NRF1、TFAM단백표체승고(P＜0.05).과표체조MMP재전염24 h후교대조조승고2.313％(t=5.546,P=0.005),48 h후승고14.872％(t=8.537,P=0.001).선립체합성지표상대mtDNA고패수,재전염24 h후량조간차이무통계학의의(P＞0.05),48 h후과표체조명현고우대조조(t=5.760,P=0.005);세포ATP함량재전염후24 h량조간차이무통계학의의(P＞0.05),48 h후과표체조ATP함량교대조조명현승고(t=22.040,P=0.008).결론 과표체Nrf2가이촉진흑소세포선립체생물합성상관기인화단백적표체,진이촉진선립체생물합성,상조선립체공능상관적지표.
Objective To explore the effects of NF-E2-related factor 2 (Nrf2) overexpression on mitochondrial biosynthesis and function in melanocytes.Methods An immortalized human vitiligo melanocyte cell line PIG3V was used in this study.An overexpression plasmid Nrf2-pEX-1 containing the full-length Nrf2 gene was constructed.PIG3V cells were divided into 3 groups:blank group receiving no treatment,control group transfected with the pEX-1 plasmid,overexpression group transfected with the Nrf2-pEX-1 plasmid.After transfection,real-time quantitative reverse transcription-PCR (RT-PCR) and Western blot were performed to determine the mRNA and protein levels of mitochondrial biosynthesis-related factors (including Nrf2,nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM)) respectively;RT-PCR was also conducted to measure the copy number of mitochondrial DNA (mtDNA),and flow cytometry to estimate mitochondial membrane potential (MMP);luciferase reporter system was used to estimate the intracellular adenosine triphosphate (ATP) level.Statistical analysis was carried out by using a two-sample t-test.Results After transfection,a significant increase was observed in the mRNA expression levels of Nrf2 and NRF1 at 24 hours (both P ＜ 0.001) and in those of Nrf2 and TFAM at 48 hours (both P ＜ 0.05),but no significant change was noted in the mRNA expression level of TFAM at 24 hours (P ＞ 0.05) or in that of NRF1 at 48 hours (P ＞0.05) in the overexpression group compared with the control group.In the case of Nrf2,NRF1 and TFAM protein levels,the overexpression group showed significant increases compared with the control group at 48 hours after transfection (all P ＜ 0.05),while no significant difference was noted between the two groups at 24 hours.Compared with the control group,MMP in the overexpression group increased by 2.313％ at 24 hours (t =5.546,P =0.005) and by 14.872％ at 48 hours (t =8.537,P =0.001) after transfection.Both the relative copy number of mtDNA and ATP level were similar between the overexpression group and control group at 24 hours after transfection (both P ＞ 0.05),but significantly higher in the overexpression group than in the control group at 48 hours (t =5.760,P =0.005;t =22.040,P =0.008).Conclusion Up-regulation of Nrf2 pathway can improve mitochondrial function and biosynthesis in PIG3V cells likely by promoting the expressions of mitochondrial biosynthesis-related genes and proteins.