糖尿病性白内障大鼠与正常大鼠晶状体蛋白质组差异分析

发布时间：2018-03-19 01:03

本文选题：蛋白质组学　切入点：晶状体　出处：《吉林大学》2011年硕士论文　论文类型：学位论文

【摘要】：糖尿病性白内障是一种常见的糖尿病并发症,在糖尿病引起的眼病中位居第二位,其发病机制有诸如：渗透压学说、蛋白质糖基化学说、氧化应力学说等。由于晶状体是生物体内蛋白质含量最高的组织,很适于应用蛋白质组学的方法来对其进行研究。近年来,随着蛋白质组学技术的建立和发展,该技术已越来越多的应用到晶状体疾病的研究中。蛋白质组学是从蛋白质组的水平上认识生命活动的机理和疾病发生的机制,它是对研究对象蛋白质水平整体的研究,其中双向电泳技术是蛋白质组学的核心技术,它是截至目前为止常用的能连续在一块胶上分离数千种蛋白质的唯一方法。目的本实验应用差异凝胶电泳的方法(2-D DIGE)结合图像分析软件找出混浊程度不同的糖尿病性白内障大鼠晶状体与正常大鼠晶状体蛋白质总体变化趋势,旨在发现有差异表达的蛋白质,为质谱分析和确定在疾病发生过程中起主要作用的蛋白质打下基础。方法运用小剂量链脲佐菌素(STZ)+特殊膳食喂养法饲养同一周龄的SD大鼠20只,每组10只,另取10只同周龄SD大鼠作对照组。后分别取造模后6周、20周及对照组大鼠晶状体各10只作为样本。所有样品经过研磨碎裂,使用Clean-up试剂盒除盐,及运用蛋白质试剂盒蛋白定量；再从每样品中取出50μg,用荧光染料Cy3、Cy5标记所选的蛋白质样品,内标用Cy2染料标记；然后混匀每块胶中的样品,将样品加入固相pH梯度干胶条内,进行等电聚焦(IFE)；等电聚焦结束后,需平衡胶条2次,然后转移至SDS-PAGE凝胶中进行二向电泳(SDS—PAGE);结束后,采用激光扫描仪进行荧光扫描,并使用DeCyder差异分析软件分析,找出差异表达蛋白质点。结果最终三组样品共检测到65个有差异蛋白质点,随晶状体混浊程度的加重,可见39个点上调表达,26个点下调表达(蛋白表达量上调或下调超过50%即1.5倍、p0.05的蛋白点定义为差异表达的蛋白质点),其中差异量达到3倍的点共19个(P0.05)。结论糖尿病性白内障大鼠和正常大鼠晶状体存在蛋白表达的差异,糖尿病性白内障大鼠混浊程度不同的晶状体和正常大鼠晶状体同一匹配的蛋白存在表达量的上升或下降。这种蛋白质的差异变化可能与糖尿病性白内障的发病机制有关。
[Abstract]:Diabetic cataract is a common diabetic complication. It is the second most common diabetic eye disease. Its pathogenesis includes osmotic pressure theory, protein glycosylated chemistry, The theory of oxidative stress. Because lens is the tissue with the highest protein content in organism, it is very suitable to use proteomics method to study it. In recent years, with the establishment and development of proteomics technology, This technique has been applied more and more to the study of lens diseases. Proteomics is the understanding of the mechanism of life activity and the mechanism of disease from the proteome level. It is the whole study of the protein level of the object of study, in which two-dimensional electrophoresis is the core technology of proteomics. It is the only method used so far to separate thousands of proteins on a piece of glue. Purpose. In this experiment, we used differential gel electrophoresis (DIGE) and image analysis software to find out the general change trend of lens protein in diabetic cataract rats with different turbidity degree and normal rats. The aim of this study is to find differentially expressed proteins and to lay a foundation for mass spectrometry analysis and identification of proteins that play a major role in the pathogenesis of disease. Method. Twenty Sprague-Dawley rats of the same week age were fed with a special diet with low dose streptozotocin (STZ) for 10 rats in each group. Another 10 SD rats of the same age were taken as control group. After 6 weeks and 20 weeks of model making and 10 rats of control group, the lens of each group were taken as samples. All the samples were ground to pieces, Clean-up kit was used to desalinate and protein kit was used to quantify protein. Then 50 渭 g of each sample was taken out, the selected protein sample was labeled with fluorescent dye Cy3OC5, and the internal standard was labeled with Cy2 dye. Then, the samples in each gel were mixed together, and the samples were added into the solid phase pH gradient dry tape for isoelectric focusing. We need to balance the tape twice, then transfer it to SDS-PAGE gel for two dimensional electrophoresis. After that, we use laser scanner for fluorescence scanning and DeCyder differential analysis software to find the differentially expressed protein spots. Results. In the end, 65 differentially expressed protein spots were detected in the three groups, with the increase of lens opacity. The results showed that 39 sites up-regulated and 26 down-regulated (protein expression more than 50%, i.e. 1.5-fold p0.05) were defined as differentially expressed protein spots. Conclusion. There was a difference in protein expression between diabetic cataract rats and normal rats. The expression of the same matching protein in the lens of diabetic cataract rats with different degree of opacity was increased or decreased, which may be related to the pathogenesis of diabetic cataract.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R776.1

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