【作者】 史泽雅； 霍怡彤； 侯佳楠； 王璐； 王晨阳； 余婧婧； 王文桃； 张锐虎； 陈朝阳；

【Author】 SHI Zeya;HUO Yitong;HOU Jianan;WANG Lu;WANG Chenyang;YU Jingjing;WANG Wentao;ZHANG Ruihu;CHEN Zhaoyang;Laboratory Animal Center of Shanxi Medical University,Shanxi Key Laboratory of Experimental Animal Science and Animal Model of Human Disease;

【机构】 山西医科大学实验动物中心山西省实验动物与人类疾病动物模型重点实验室；

【摘要】 目的通过选择性繁殖定向培育建立近交系中国地鼠自发性2型糖尿病动物模型并对该模型进行评价,然后基于蛋白质组学和代谢组学对其发病机制进行初步研究。方法根据中国地鼠空腹血糖(FBG)和餐后血糖(PBG)值,选取对照组(FBG≤4.5 mmol/L且PBG<6.0 mmol/L)与糖尿病组(FBG≥6.0 mmol/L且PBG>7.0 mmol/L),测定动物糖尿病基础病理生理特征及Leptin,Adiponectin,Pparg,Glut4和Akt基因在肝脏与骨骼肌组中的表达。应用基于质谱的蛋白质组学与代谢组学分析中国地鼠小肠与肝脏组织的差异表达蛋白与血清中的差异代谢物,利用生物信息学工具进行分析。并且利用Western Blotting和RT-qPCR验证小肠与肝脏组织中各自5个候选差异表达蛋白表达水平,利用ELISA对血清中胆汁酸和花生四烯酸的含量进行检测。结果中国地鼠糖尿病组FBG和PBG中轻度增高、糖脂代谢异常、氧化应激水平升高、产生胰岛素抵抗(IR)、昼夜活动规律紊乱,胰岛、骨骼肌、肝脏组织都存在病理学改变,此外,糖脂代谢相关基因的表达也存在差异。蛋白质组学结果显示,小肠与肝脏组织中分别鉴定到213个与103个差异表达蛋白,分别富集到29条与8条KEGG通路。代谢组学结果显示,血清中共鉴定到14种差异代谢物,MPEA分析显示富集到的通路几乎均为氨基酸代谢通路。通过对小肠蛋白质组学与血清代谢组学进行联合分析,甘氨酸、丝氨酸和苏氨酸代谢途径被两组学共同富集,包括8个差异表达蛋白(PGAM1、PHGDH、PSAT1、CTH、CBS、GATM、GAMT和GCAT)与3种差异代谢物(Glycine、Homoserine和Tryptophan);对肝脏蛋白质组学的结果进行分析,发现胆汁分泌和花生四烯酸代谢两条通路显著富集,AQP9和EPHX1富集在胆汁分泌通路中表达下调,PTGES2,Cyp2c27和Cyp2c70与花生四烯酸代谢通路相关。上述候选差异蛋白的验证结果与蛋白质组学分析结果一致且糖尿病地鼠血清中胆汁酸和花生四烯酸的含量明显高于对照组。结论本课题组成功建立了近交系中国地鼠自发性2型糖尿病动物模型,且其发病特征与人类2型糖尿病非常相似,上述差异蛋白可能为IR相关表达蛋白,为探索T2DM的发病机制进行了初步研究。更多还原

【Abstract】 Objective To establish and evaluate an animal model of spontaneous type 2 diabetes in Chinese hamster inbred by selective breeding and directional breeding,and to investigate the pathogenesis of the model based on proteomics and metabolomics.Methods Control group(FBG≤4.5 mmol/L and PBG <6.0 mmol/L) and diabetes group(FBG≥6.0 mmol/L and PBG>7.0 mmol/L) were selected according to the fasting blood glucose(FBG) and postprandial blood glucose(PBG) values of Chinese hamsters.The pathological and physiological characteristics of animal diabetes mellitus and the expression of Leptin,Adiponectin,Pparg,Glut4 and Akt genes in liver and skeletal muscle were determined.Proteomics and metabolomics based on mass spectrometry were used to analyze the differentially expressed proteins in small intestine and liver tissues and the differentially expressed metabolites in serum of Hamsters.Bioinformatics tools were used for analysis.Western Blotting and RT-qPCR were used to verify the expression levels of five candidate differentially expressed proteins in small intestine and liver tissues respectively,and the contents of bile acid and arachiidonic acid in serum were detected by ELISA.Results There were slight increase of FBG and PBG,abnormal glucose and lipid metabolism,increased oxidative stress,insulin resistance(IR),disturbance of diurnal activity,pathological changes in islet,skeletal muscle and liver,and different expressions of genes related to glucose and lipid metabolism in Chinese hamsters with diabetes.Proteomic results showed that213 and 103 differentially expressed proteins were identified in small intestine and liver tissues,respectively,which were enriched into 29 KEGG pathways and 8 KEGG pathways.Metabolomics results showed that a total of 14 different metabolites were identified in serum,and MPEA analysis showed that almost all the pathways enriched were amino acid metabolic pathways.By combining small intestinal proteomics with serum metabolomics,glycine,serine,and threonine metabolic pathways were enriched by both groups.There were 8 differentially expressed proteins(PGAM1,PHGDH,PSAT1,CTH,CBS,GATM,GAMT and GCAT) and 3 differentially expressed metabolites(Glycine,Homoserine and Tryptophan).The results of liver proteomic analysis showed that the biliary secretion and arachidic acid metabolism pathways were significantly enriched,AQP9 and EPHX1 were enriched in the biliary secretion pathway,and the expression of PTGES2,Cyp2 c27 and Cyp2 c70 were correlated with the arachidic acid metabolism pathway.The verification results of the above candidate differential proteins were consistent with the results of proteomic analysis,and the contents of bile acid and aracidonic acid in serum of diabetic hamsters were significantly higher than those of the control group.Conclusion Our research group has successfully established the animal model of spontaneous type 2 diabetes in Chinese hamsters,and its pathogenesis characteristics are very similar to human type 2 diabetes.The above differential proteins may be IR-related expression proteins,and a preliminary study was conducted to explore the pathogenesis of T2DM.更多还原