不同渗透压条件下金钱鱼肾原代细胞的差异蛋白分析

Differential proteins analysis for primary renal cells of Scatophagus argus under different osmotic stresses

为了研究海水鱼类肾脏的渗透压调节功能,以金钱鱼肾原代细胞作为研究对象,使用低渗、等渗和高渗培养基处理细胞24 h,通过Label-free定量法进行蛋白质组学分析,质谱定性得到蛋白质共计3 787个,低渗组与等渗组比较,得到显著差异蛋白14个;高渗组与等渗组比较,得到显著差异蛋白31个。筛选标准为Ratio>+/-2,且P <0. 05。对差异表达蛋白进行Gene Ontology(GO)功能注释,GO富集分析以及蛋白质聚类分析,并挑选了与细胞骨架、能量合成以及蛋白结合相关的蛋白质进行了mRNA水平的定量检测,从基因和蛋白水平了解肾脏细胞在渗透调节中的变化。结果显示:低渗组中,细胞骨架相关蛋白质受到的影响较大,其中包括微管蛋白α-4A链、微管蛋白β链、肌球蛋白-11以及typeⅠcytoskeletal 18-like,而高渗组中差异显著的蛋白质多为细胞外间质蛋白,如纤连蛋白、胶原蛋白等。因此推测低渗刺激后,胞内渗透压高于胞外渗透压,细胞立即膨胀并大量表达细胞骨架蛋白应对低渗刺激,适应细胞形态的改变;而高渗刺激下,细胞处于失水状态,形态骤缩,影响细胞间的粘连,而fn、plod2和arf1升高表明,细胞正在修复受损蛋白。本研究从细胞水平初步分析了金钱鱼肾脏在不同渗透压刺激下的应答机制,为研究细胞水平的渗透压调节机制提供了新的观点。

In order to study the osmoregularatory function of kidney in Scatophagus argus,primary renal cells were treated with hyposmotic,isosmotic and hyperosmotic media for 24h.The label-free quantitative method was employed in proteomic analysis,and a total of 3,787proteins were obtained by mass spectrometry. Compared with isotonic group,14proteins were significantly expressed in the hypotonic group and 31 in the hyperosmotic group,respectively (Ratio >+/-2and P <0.05).Differential expression profiles were analyzed by Gene Ontology (GO)annotations,enrichment analysis and clustering analysis.The proteins associated with cytoskeleton,energy synthesis and protein binding were selected for quantitative detection at mRNA level to confirm changes of renal cells at gene level and protein level.In hyposmotic group, cytoskeleton-related proteins were up-expressed significantly,including tubulin alpha-4A chain,tubulin beta chain,myosin-11and type I cytoskeletal 18-like.In the hyperosmotic group,significantly expressed proteins were of extracellular matrix,such as fibronectin and collagen.Therefore,it is assumed that after hypo-osmotic stimulation,intracellular osmotic pressure was higher than extracellular osmotic pressure,and the cells expanded immediately,then expressed cytoskeleton related proteins in large quantities,thus affecting other ion channels.Under hyper-osmotic stimulation,the cells were in a state of water loss and morphologic contraction,which affected the adhesion between cells.However,the increase of fn and plod2 showed that the cells were repairing the damaged protein.This study showed changes of protein expression after anisotonic shocks of primary renal cells in Scatophagus argus and may provide references for further studies of osmoregulation.