权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Proteomic analysis of lifestyle related diseases

生活方式相关疾病的蛋白质组学分析

基本信息

批准号：
16590039
负责人：
MATSUNAGA Hisami
金额：
$ 2.05万
依托单位：
Mukogawa Women's University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

In the last few years, the interest in exploring the proteome of biological fluids has expanded through the search for biomarkers of lifestyle related diseases. In this study, we investigated the proteome of pancreas in an animal model of metabolic syndrome, SHR/NDmcr-cp rat (SHR/NDmcr-cp). The pancreas tissues extirpated from twenty-week-old male SHR/NDmcr-cp and normative rats, Wistar-Kyoto/Izm (WKY/Izm). These two samples (1 g wet weight) were homogenized with 10 mL of a lysis buffer, containing of 7 M urea, 4% 3-[(cholamidopropyl) dimethyl ammonio]-1-propanesulfonate, 2 M thiourea, and 5 mM magnesium acetate / 30 mM Tris-HC1 buffer (pH 8.0). After centrifugation at 400,000 x g for 60 min at 4 0C, the supernatant was used for a two-dimensional electrophoresis (2-DE) sample. One hundred μg of a protein was diluted to 350 μL with a rehydration solution and applied onto Immobiline dry strips (linear gradient between pH 3 and 10, 13 cm (GE Healthcare Bio-sciences, NJ, USA)). The protein … More s were focused on a IPG-phor system (GE Healthcare Bio-sciences). After the first dimension electrophoresis, the strips were equilibrated for 15 min in a solution containing dithiothreitol, which was replaced by iodoacetamide. Separation in the second dimensional electrophoresis was carried out on a SE 600 Ruby (GE Healthcare Bio-sciences) using a 12.5% SDS-polyacrylamide gel. Pancreas proteins separated on a 2-DE gel were stained with coomassie brilliant blue. The 32 spots of the SHR/NDmcr-cp sample on the 2-DE gel were more stained than those of the WKY/Izm sample. Then those 32 spots were cut, and soaked with digestion buffer containing trypsin. After the overnight protein digestion, peptide fragments were desalted by Zip tips C18, extracted and analyzed with AXIMA-CFR plus MALDI TOF-MS. Thereafter, the protein was identified by a MASCOT program using Swiss-Prot and NCBInr databases limited to Rodents species. Ten proteins provided a significant match with databases' proteins. Those proteins included a-enolase, phosphoglycerate kinase, branched-chain-amino acid transferase, carboxy-peptidase Al precursor, α-protein, inositol monophosphatase chymotrypsinogen B precursor, hypoxanthine-guanine phosphoribosyltransferase, nucleoside diphosphate kinase B, elastase-2 precursor and annexin A5. Further study for the proteome of plasma in an animal model of metabolic syndrome is ongoing in order to search for biomarkers of lifestyle related diseases. Less

在过去的几年中，通过寻找生活方式相关疾病的生物标志物，对探索生物液体蛋白质组的兴趣已经扩大。本研究对代谢综合征动物模型SHR/NDmcr-cp大鼠（SHR/NDmcr-cp）胰腺组织蛋白质组进行了研究。取20周龄雄性SHR/NDmcr-cp和正常Wistar-Kyoto/Izm（WKY/Izm）大鼠胰腺组织。将这两个样品（1 g湿重）用10 mL裂解缓冲液均质化，所述裂解缓冲液含有7 M尿素、4% 3-[（胆酰胺丙基）二甲基铵基]-1-丙磺酸盐、2 M硫脲和5 mM乙酸镁/ 30 mM Tris-HCl缓冲液（pH 8.0）。在4 ℃下以400，000 × g离心60分钟后，将上清液用于二维电泳（2-DE）样品。用再水化溶液将100 μg蛋白质稀释至350 μL，并应用于Immobiline干条（pH 3至10之间的线性梯度，13 cm（GE Healthcare Bio-sciences，新泽西州，美国）。蛋白质 ...更多信息的研究集中在IPG-phor系统（GE Healthcare Bio-sciences）上。在第一维电泳后，将条带在含有二硫苏糖醇的溶液中平衡15分钟，所述二硫苏糖醇被碘乙酰胺代替。在SE 600 Ruby（GE Healthcare Bio-sciences）上使用12.5%SDS-聚丙烯酰胺凝胶进行第二维电泳中的分离。用考马斯亮蓝对分离的胰腺蛋白进行染色。2-DE凝胶上SHR/NDmcr-cp样品的32个斑点比WKY/Izm样品的染色程度更深。然后将这32个点切下，用含有胰蛋白酶的消化缓冲液浸泡。过夜蛋白质消化后，通过Zip tips C18脱盐肽片段，提取并用AXIMA-CFR plus MALDI TOF-MS分析。此后，通过MASCOT程序使用限于啮齿动物物种的Swiss-Prot和NCBInr数据库鉴定蛋白质。10种蛋白质与数据库的蛋白质提供了显著匹配。这些蛋白质包括α-烯醇化酶、磷酸甘油酸激酶、支链氨基酸转移酶、羧肽酶Al前体、α-蛋白、肌醇单磷酸酶胰凝乳蛋白酶原B前体、次黄嘌呤鸟嘌呤磷酸核糖基转移酶、核苷二磷酸激酶B、弹性蛋白酶-2前体和膜联蛋白A5。为了寻找生活方式相关疾病的生物标志物，正在对代谢综合征动物模型中的血浆蛋白质组进行进一步研究。少