Objective To investigate the effect of zinc-α2-glycoprotein(ZAG)on the expression of relevant factors of mitochondria biosynthesis in adipocytes.Methods The total RNA was extracted from mouse liver cells and the sequence of ZAG was amplified with RT-PCR.The PCR product was cloned into the eukaryocyte expression vector pcDNA3.1 (-) after enzyming by Xba Ⅰ and Hind Ⅲ.After identification by double restriction enzyme digestion and DNA sequencing,the recombinant eukaryotic expression plasmid pcDNA3.1 (-)-mZAG was transfected into 3T3-L1 cells by lipofectamine 2000.The stable transfected 3T3-L1 cell line was established after selection with G418.After 24 hours of transfection,the rnRNA and protein expressions of PGC-1α,NRF-1,NRF-2,and mtTFA were detected by RT-PCR and Western blot respectively.Results The pcDNA3.1 (-)-mZAG plasmid was constructed and transfected into the 3T3-L1 cells successfully.Compared with the untransfected group and pcDNA3.1(-) transfected group,the level of mRNA and protein expressions of PGC-1α,NRF-1,NRF-2,and mtTFA were significantly up-regulated in the pcDNA3.1 (-)-mZAG transfected group (P＜0.01).Conclusion The recombinant eukaryotic expression vector pcDNA3.1 (-)-mZAG and 3T3-L1 cell line stable over-expression of ZAG are established.ZAG promotes mRNA and protein expressions of rnitochondria biogenesis genes.%目的 探讨锌α2糖蛋白(ZAG)对脂肪细胞线粒体生物合成相关因子mRNA和蛋白表达的影响. 方法 构建重组小鼠ZAG真核表达质粒pcDNA3.1(-)-mZAG,经脂质体转染3T3-L1细胞,24 h后提取RNA和蛋白质.RT-PCR法及Western blot法检测过氧化物酶增殖型受体7辅助活化因子1α(PGC-1α)、核呼吸因子1(NRF-1)、核呼吸因子2(NRF-2)、线粒体转录因子A(mtTFA) mRNA和蛋白的表达. 结果 与未转染组和pcDNA3.1(-)组比较,pcDNA3.1(-)-mZAG组3T3-L1细胞PGC-1α、NRF-1、NRF-2、mtTFA的mRNA和蛋白表达均上调(P＜0.01). 结论 ZAG对脂肪细胞线粒体生物合成相关因子PGC-1α、NRF-1,NRF-2、mtTFA的mRNA和蛋白表达均有促进作用.
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