【摘要】 目的:研究Cox7a2的荧光表达载体在TM3睾丸Leydig细胞中的表达和定位及对ERK1/2磷酸化的影响。方法:将Cox7a2克隆到pEYFP-N1。细胞转染和细胞荧光显微镜观察其在TM3细胞中的表达和定位,Westernblot检测ERK1/2磷酸化水平。结果:在TM3细胞中,绿色荧光YFP-Cox7a2和红色荧光线粒体标记物-Mitotracker有完全的共定位,过量表达YFP-Cox7a2能引起ERK1/2磷酸化水平增高。结论:成功表达了YFP-Cox7a2融合蛋白,确认了其在TM3细胞的线粒体定位。YFP-Cox7a2过表达和ERK1/2磷酸化水平有联系,为探讨Cox7a2对TM3细胞睾酮分泌及其相关信号传导通路的影响奠定了基础。更多还原

【Abstract】 Objective:To study the expression, localization and its relationship with ERK1/2 phosphorylation of Cox7a2 by construction of cox7a2-pEYFP-N1 in TM3 mouse Leydig cells.Methods:Cox7a2 was cloned into pEYFP-N1/2 between BglⅡ and EcoRⅠsites. Cell-transfection and living-cell fluorescence imaging microscopy were employed to investigate the sub-cellular localization of YFP-Cox7a2. Western blotting was used to test the phosphrylation of ERK1/2.Results:Cox7a2 green fluorescence protein was well co-localized with red fluorescence mitochondrion marker-Mitotracker in TM3 mouse Leydig cells. Over-expression of YFP-Cox7a2 increased ERK1/2 phosphorylation in TM3 cells.Conclusions:The fluorescent expression vector of Cox7a2 was constructed successfully and YFP-Cox7a2 was expressed and located in mitochondria. Over-expression of YFP-Cox7a2 in TM3 mouse Leydig cells may be related with activation of ERK signaling pathway, which could provide useful clue for further research on androgen biosynthesis dysfunction in aging male.更多还原