草酸和草酸钙结晶损伤肾小管上皮细胞的差异蛋白质组学研究

Comparative proteomic analysis of renal tubular epithelial cell injury caused by oxalic acid and calcium oxalate monohydrate

目的分析并鉴定草酸和一水草酸钙（COM）结晶损伤人肾小管上皮细胞（HK2）后细胞蛋白质谱的表达变化，探讨肾小管上皮细胞受损在肾结石形成中的可能作用。方法体外培养正常HK2细胞至90％融合后，换无血清培养基，随机分为2组。实验组培养基内加入2mmol／L草酸＋200mg／LCOM结晶，37℃孵育。分别抽提2组细胞的总蛋白，双向凝胶电泳结合液相色谱-电喷雾离子阱质谱（LC—ESI—MS／MS）技术对2组中差异表达的蛋白质进行分离和鉴定。蛋白印迹法对鉴定出的蛋白进行验证。结果成功建立细胞总蛋白的双向凝胶电泳图谱，经软件分析和质谱鉴定出差异蛋白质12个：FK506结合蛋白4、α-烯醇酶、M2型丙酮酸激酶、ATP合成酶α亚单位、3’，5，-二磷酸核苷酸酶1、核磷蛋白2、L-乳酸脱氢酶B、芽胞发芽蛋白3、Cofilin-1、Fascin、40S核糖体蛋白S17和胞液氨基肽酶1。差异蛋白涉及细胞能量代谢、细胞增殖、凋亡、钙离子通道活性调控、细胞运动及信号转导等多种生理活动。蛋白印迹法检测示HK-2细胞损伤后EN01蛋白表达上调，而Cofilin-1表达下调，证实双向凝胶电泳和质谱分析可靠。结论高浓度草酸和COM结晶可使正常人HK-2细胞蛋白表达发生改变，这些差异表达蛋白既可起到细胞的自我保护作用，又可能通过相应途径在肾结石的形成中起重要作用。

Objective To analyze and identify the differentially expressed proteins in human renal tubular epithelial ceils （HK-2） after injury caused by oxalic acid and calcium oxalate monohydrate （COM） crystal, and to explore the potential role of renal tubular cell injury in kidney stone formation. Methods Normal HK-2 cells were cultured in vitro and the culture medium was changed with serumfree medium after cell growth to confluence. Oxalic acid and COM crystals （final concentration at 2 mmol/L and 200 mg/L, respectively） were added in the experimental group. Cells in both groups were then incubated at 37℃ for 12 h. The extracted proteins from both groups were separated by two dimensional electrophoresis followed by analysis, and the differentially expressed proteins were identi- fied by liquid chromatography electrospray ionization tandem mass spectrometry （LC-ESI-MS/MS）. Two identified proteins were then verified by western blot. Results Reproducible two dimensional gel images of the proteins from both groups were successfully obtained. By using LC-ESI-MS/MS, 12 proteins： FKS06-binding protein 4, isoform alpha enolase of alpha-enolase, isoform M1 of pyruvate kinase isozymes M1/M2, ATP synthase subunit alpha, isoform 1 of 3＇ （2＇）, 5＇-bisphosphate nucleotidase 1, isoform 2 of nucleophosmin, L-lactate dehydrogenase B chain, Budding uninhibited by benzimidazoles 3, Cofilin-1, Fascin, pyIsoform 1 of cytosol aminopeptidase, were identified. The deferential- ly expressed proteins were related to cellular processes including energy metabolism, cell multiplica- tion, apoptosis, Ca2＋ channel activity regulation, cell movement and signal transduction. Western blot verified that higher ENO1 but lower Cofilin-1 expressed in HK-2 cells after the injury. Conclusions High level oxalic acid and COM crystals can cause protein expression profile changes in normal human HK-2 cells. The changes of protein expression may not only protect HK-2 cells from being injured, but also be related to kidney stone formation.