摘要
目的 筛选及鉴定糖尿病大鼠海马纹状体区的差异蛋白表达.方法 采用随机方法,将SD大鼠分糖尿病组(n=15)及正常组(n=10).采用高脂高糖饮食联合一次性腹腔注射链脲佐菌素的方法制备糖尿病大鼠模型,以糖尿病大鼠及正常SD大鼠海马纹状体区为研究对象,采用核素相对标记与绝对定量技术(iTRAQ)联合双向液相色谱-串联质谱(2DLC-MS/MS)鉴定糖尿病大鼠海马纹状体区差异表达蛋白,对所获蛋白行生物信息学分析,筛选其中几个候选蛋白,行Western印迹检测,验证这些蛋白在糖尿病大鼠海马纹状体区中的表达.结果 成功建立糖尿病模型,共鉴定出347种差异表达蛋白(其中上调蛋白139个,下调蛋白208个).包括与帕金森相关的铁蛋白代谢型谷氨酸受体2型,铁蛋白,亮氨酸重复序列蛋白及Complexin2蛋白等.KEEN检索提示多巴胺能神经突触路径,补体通路,缝隙连接,自噬路径及FcγR介导的吞噬作用等通路呈现显著的差异蛋白富集.蛋白质功能相互作用网状图发现铁蛋白(ferrin)、醛酸/酮还原酶(aldehyde/ketone reductases,ARK),钙结合蛋白(Ca2+-binding protein,CaBP),蛋白酪氨酸磷酸酶(protein tyrosine phosphatase,PTP)等蛋白处于相互作用网络的交叉点.筛选上述4个蛋白后运用Western印迹验证,发现这4种蛋白表达水平与质谱结果一致(P<0.05).结论 采用iTRAQ结合2DLC-MS/MS技术,能有效地筛选糖尿病大鼠海马纹状体区差异蛋白的表达,为糖尿病对中枢神经系统损伤机制研究提供新线索,对神经退行性疾病的诊断及治疗提供新思路及潜在的治疗靶点.
Objective To screen the deferentially expressed proteins in hippocampus and striatum in rat models of diabetes mellitus and normal SD rats,and to elucidate the effects of hyperglycemia on central nervous system.Methods SD rats were randomly divided into normal group(n=10)and hyperglycemia group(n=15);rat models of diabetes were induced by the high-fat and high-sugar diet combined with single intraperitoneal injection of streptozotocin.The hippocampus and striatum tissues from hyperglycemia rat and normal SD rat(control group)were selected as specimens.Isobaric tags for relative and absolute quantification(iTRAQ)technique and 2-dimensional liquid chromatography-tandem mass spectrometry(2D-LC-MS/MS)were used for identifying the deferentially expressed proteins.The deferentially expressed proteins were analyzed by bio-informatics for searching candidate proteins.Finally,the candidate proteins were verified by Western blotting.Results A total of 347 proteins showed significantly different expressions,which included 139 up-regulated proteins and 289 down-regulated proteins.These proteins included metabolic glutamate receptor 2,ferritin,leucine repeat protein and complex 2,which are related to Parkinson′s disease.KEEN search indicated that the pathway of dopaminergic synaptic,complement,gap junction,autophagic and Fcgamma R-mediated phagocytosis were involved with these significant differentially expressed proteins.The protein interaction network showed that ferrin,aldehyde/ketone reductase(ARK),Ca2+-binding protein(CaBP),and protein tyrosine phosphatase(PTP)were located at the crossed nodes.The expression levels of ferrin,ARK,PTP,and CaBP,which were verified by Western blotting,and the results were consistent with those of the quantitative mass spectrometry(P<0.05).Conclusion Deferentially expressed proteins in hippocampus striatum of diabetic rats can be effectively screened by iTRAQ technique combined with 2D-LC-MS/MS.It may provide new clues for the mechanism of neurodegenerative disease.
作者
买尔哈巴
迪力木拉提·阿不来提
杨新玲
贾玉敏
耿飞飞
Maierhaba null;Dilmurat Ablat;Yang Xinling;Jia Yumin;Geng Feifei(Fourth Branch of Comprehensive of Department of Internal Medicine,The First Teaching Hospital of Xinjiang Medical University,Urumqi 830054,China;First Aid Centre,People′s Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China;Department of Neurology,The Second Teaching Hospital of Xinjiang Medical University,Urumqi 830054,China)
出处
《中华内分泌代谢杂志》
CAS
CSCD
北大核心
2019年第11期973-980,共8页
Chinese Journal of Endocrinology and Metabolism
基金
新疆维吾尔自治区自然科学基金项目(2015211C060)。
关键词
糖尿病
差异蛋白组学
核素相对标记与绝对定量技术
双向液相色谱-串联质谱
Diabetes mellitus
Proteomics analysis
Isobaric tags for relative and absolute quantification
2-dimensional liquid chromatography-tandem mass spectrometry
