人钠/二羧酸协同转运蛋白3基因调控肾小管上皮细胞线粒体膜电位的研究

Regulation of mitochondrial membrane potential by human Na^+/dicarboxylate cotransporter 3 in human proximal tubular epithelial cells

目的 观察人钠/二羧酸协同转运蛋白3(hNaDC3)对人肾脏近曲小管上皮细胞(HKC)线粒体膜电位的变化及其对细胞能量代谢的影响。方法 应用亚克隆技术构建正义pcDNA3-hNaDC3和反义pcDNA3-AhNaDC3两个真核表达载体,通过脂质体LipofectAMINE将pcDNA3-hNaDC3及pcDNA3-AhNaDC3转染至HKC细胞。克隆筛选后,用RT-PCR、Northern印迹及Western印迹鉴定外源基因的整合和表达。荧光探针JC-1观察各细胞系线粒体膜电位的变化。结果 外源hNaDC3基因稳定整合到HKC细胞基因组中,并获得高、低表达。转染正义hNaDC3 cDNA的HKC细胞线粒体膜电位降低,JC-1在线粒体内形成单体,发出绿色荧光;而转染反义hNaDC3 cDNA的HKC细胞线粒体膜电位略微升高,JC-1形成聚合体,发出红色荧光。结论 hNaDC3过表达引起线粒体膜电位降低,反义hNaDC3则使线粒体膜电位略微升高。提示NaDC3可能通过使线粒体膜电位下降,参与了细胞能量代谢。

Objective To investigate the effect of sense and antisense hNaDC3 on mitochondrial membrane potential and energy metabolism of the cell in human proximal tubular epithelial cells(HKC). Methods Two recombinant eukaryotic expression vectors,pcDNA3-hNaDC3 encoding sense mRNA and pcDNA3-AhNaDC3 encoding antisense mRNA were constructed by using DNA recombining techniques. These two vectors were then transfected into HKC with LipofectAMINE. RT-PCR,Northern blotting and Western blotting were used to detect the integration and expression of hNaDC3. Fluorescent JC-1 in combination with confocal fluorescence microscopy and flow cytometry was used to detect the changes of in normal HKC,pcDNA3 transfected-,pcDNA3-hNaDC3 transfected- and pcDNA3-AhNaDC3 transfected-HKC.Results Both sense and antisense hNaDC3 were successfully integrated into HKC and expressed the sense and antisense hNaDC3 RNA. Compared with control,HKC transfected with pcDNA3-hNaDC3 had a low ,JC-1 monomer was formed in the inner membrane of mitochondria and emitted green fluorescence, while HKC transfected with pcDNA3-AhNaDC3 had a little high ,JC-1 monomer was associated to aggregates,which emitted red fluorescence. Conclusions Overexpression of hNaDC3 induced by sense hNaDC3 causes the decrease of ,in contrast,suppression of hNaDC3 expression by antisense hNaDC3 causes the increase of . NaDC3 may be involved in energy metabolism of the cell by causing the decrease of mitochondrial membrane potential.