高糖培养下人肾小球系膜细胞蛋白表达谱变化的研究

Protein expression profile of human glomerular mesangial cells under high glucose

目的 利用比较蛋白质组学双向电泳技术研究体系,观察高糖培养下人肾小球系膜细胞（HMC）蛋白表达谱的变化.方法 人肾小球系膜细胞分为高糖培养组（30 mmol/L）和正常糖组（5 mmol/L）,培养48 h后收集细胞,提取总蛋白,以DIGE饱和荧光标记,双向荧光差异凝胶电泳.应用Typhoon多功能成像系统扫描凝胶,DeCyder 2-D差异分析软件进行图像分析,寻找差异表达蛋白质.采用基质辅助激光解析电离飞行时间质谱（MALDI-TOF-MS）和蛋白质数据库检索鉴定差异蛋白.结果 通过DeCyder 2-D差异分析软件,发现正常糖组和高糖培养组之间差异大于1.5倍的蛋白质斑点147个,对其中96个差异蛋白质斑点进行了肽质指纹图分析,鉴定出37种蛋白质.其中磷脂酰乙醇胺结合蛋白1（PEBP-1）、粒溶素、ATP合成酶H＋转运线粒体F0复合体亚基F2仅在高糖组表达.高糖刺激后表达上调的蛋白质有24个,包括嗜酸细胞阳离子蛋白、RGS膜相互作用蛋白16（MIR16）、肽酰-脯氨酰-顺反式异构酶、disks large homolog DLG2、早发性乳腺癌2（BRCA2）、儿茶酚-邻-甲基转移酶等;表达下调的蛋白有5个,包括O-GlcNAc transferase-interacting protein 106 000 isoform 1、proteasome beta 6 subunit precursor、NEFA-interacting nuclear protein NIP30等.结论 高糖培养下HMC内147种蛋白质的表达发生变化.这些蛋白质广泛参与高糖对HMC的细胞骨架、糖代谢、细胞分裂、基因转录、信号转导、磷酸化、细胞增殖、凋亡等的调节.深入分析这些差异表达蛋白的功能与调控,有望为阐明糖尿病肾病的发病机制提供重要的实验依据.

Objective To analyze the protein expression profile of human glomerular mesangial cells （HMCs） under high glucose and to characterize molecular functions and biological processes. Methods HMCs were divided into high glucose cultured group （30 mmol/L） and normal glucose cultured group （5 mmol/L）. The total proteins were extracted after culture for 48 hours. The total proteins of the two groups were separated using two-dimensional fluorescence difference in gel electrophoresis （2-D DIGE） and analyzed using DeCyder 2-D difference analysis software. The differentially expressed proteins were further identified using in-gel digestion with trypsin, of which peptide extracts were prepared for MALDI-TOF-MS analysis. Protein identifications were searched in the NCBI protein database using the Mascot search engine. Results One hundred and forty-seven protein spots whose expression levels were significantly increased or decreased more than 1.5 folds under high glucose were identified. Ninety-six differentially expression protein spots were analyzed by peptide mass fingerprinting and 37 kinds of proteins were identified. The protein spots of phosphatidylethanolamine binding protein 1 （PEBP-1）, granulysin,ATP synthase H ＋ transporting mitochondrial FO complex subunit F2 were observed only in high glucose group. The expression of 24 proteins was up-regulated by high glucose, including eosinophil cationic protein, RGS membrane-interacting proteins 16 （MIR16）, peptidyl-prolyl cis-trans isomerase, disks large homolog DLG2, breast cancer 2, early onset （BRCA2）, Catechol-O-methyltransferase etc. The expression of 5 proteins was down-regulated by high glucose, including O-GlcNAc transferase-interacting protein 106 000 isoform 1, proteasome beta 6 subunit precursor,NEFA-interacting nuclear protein NIP30 etc. Conclusions Expression of 147 proteins in HMCs alters under high glucose. These proteins are involved in the regulation of cytoskeleton, glucose metabolism, cell division, gene transcription, signal transduction, phosphorylation, cell proliferation,apoptosis etc. In-depth analysis of these differentially expressed proteins＇ function and crosstalk is expected to provide an important experimental basis for clarifying the pathogenesis of diabetic nephropathy.