原核表达的萝卜PHGPx和谷胱甘肽对NIH3T3细胞氧化损伤的联合保护作用  

Protection Effects of Prokaryotic Expressed Radish Phospholipid Hydroperoxide Glutathione Peroxidase and Glutathione on Hydroperoxide-mediated Injury in Mouse NIH3T3 Fibroblasts

作　　者：刘冠兰[1] 李甜[1] 刘进元[1] 严泽民[2] 段明星[1] 

机构地区：[1]清华大学生命科学学院,北京100084 [2]江苏隆力奇生物科技股份有限公司,常熟215555

出　　处：《中国生物工程杂志》2010年第9期13-18,共6页China Biotechnology

基　　金：国家"863"计划(2007AA100604);国家"转基因生物新品种培育"科技重大专项(2008ZX08009-003)资助项目

摘　　要：将大肠杆菌中高效表达的萝卜磷脂氢谷胱甘肽过氧化物酶(RsPHGPx)及其底物还原型谷胱甘肽(GSH)联合作用于H2O2、过氧化叔丁基(t-BHP)以及磷脂氢过氧化物(PLPCOOH)损伤的小鼠NIH3T3成纤维细胞,研究其对于细胞过氧化损伤的保护作用。发现单独加入10μg/ml RsPHGPx并不能明显保护细胞应对过氧化损伤,但是10μg/ml RsPHGPx与3mg/mlGSH共同作用,可显著提高GSH对细胞过氧化损伤的保护效果,提高细胞存活率,降低细胞膜脂质过氧化水平和胞内活性氧(ROS)水平,保护细胞维持形态完整和抑制细胞凋亡。这一联合作用结果说明RsPHGPx的催化作用可以快速清除细胞内多种过氧化物,高效地保护细胞免受过氧化损伤,为RsPHGPx的应用提供了实验依据。The radish phospholipid hydroperoxide glutathione peroxidase （RsPHGPx） fusion proteins expressed in Escherichia coli with high efficiency were purified and the antioxidant effects of RsPHGPx together with glutathione on hydroperoxide-mediated injury in mouse NIH3T3 fibroblasts were investigated.The cell viability measurement confirmed that 10μg/ml RsPHGPx,when added to the fibroblasts separately,can only slightly increase the cell viability.However,when combained with glutathione,the antioxidant effects increase dramatically.MDA assay suggests that RsPHGPx can scaveng the lipid peroxides,the results are accordance with the MTT results.ROS flourescence investigations and DAPI experiments show that RsPHGPx can increase the protection effect via inhibiting ROS genreation and this protection help maintain the normal nuclear and membrane conditions of NIH3T3 fibroblasts.The combain experiment of RsPHGPx and glutathione provides experimental evidences for the future production of RsPHGPx.

关 键 词：萝卜PHGPx 谷胱甘肽 过氧化物损伤 小鼠NIH3T3成纤维细胞 

分 类 号：Q789[生物学—分子生物学]