Andersen H U, Fey S J, Larsen P M, Nawrocki A, Hejnaes K R, Mandrup-Poulsen T, Nerup J
Steno Diabetes Center, Gentofte, Denmark.
Electrophoresis. 1997 Oct;18(11):2091-103. doi: 10.1002/elps.1150181136.
Insulin-dependent diabetes mellitus is caused by an autoimmune destruction of the beta-cells in the islets of Langerhans. The cytokine interleukin 1 inhibits insulin release and is selectively cytotoxic to beta-cells in isolated pancreatic rat islets. The antigen(s) triggering the immune response as well as the intracellular mechanisms of action of interleukin 1-mediated beta-cell cytotoxicity are unknown. However, previous studies have found an association of beta-cell destruction with alterations in protein synthesis. Thus, two-dimensional (2-D) gel electrophoresis of pancreatic islet proteins may be an important tool facilitating studies of the molecular pathogenesis of insulin-dependent diabetes mellitus. 2-D gel electrophoresis of islet proteins may lead to (i) the determination of qualitative and quantitative changes in specific islet proteins induced by cytokines, (ii) the determination of the effects of agents modulating cytokine action, and (iii) the identification of primary islet protein antigen(s) initiating the immune destruction of the beta-cells. Therefore, the aim of this study was to create databases (DB) of all reproducibly detectable protein spots on 10% and 15% acrylamide 2-D gels of neonatal rat islets (10% and 15% DB), labeled under standardized culture conditions. 1235 and 557 spots were present in 5 of 5 gels in the 15% isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) DB, respectively, whereas 995 and 378 spots were present in 5 of 5 gels in the 10% IEF and NEPHGE DB, respectively, yielding a reproducibility of spot detection between 75.2% and 91.7%. In both DBs, the average coefficient of variation of the percentage of integrated optical density (CV% of %IOD) for spots present in all gels was between 42.4% and 45.7%. When the same sample was analyzed in consecutive sets of gels on different days (interassay analysis), the average CV% of %IOD was 35.5%-36.1%. When the same sample was analyzed repeatedly in one set of gels (intra-assay analysis), the average CV% of %IOD was 30.2% in the IEF gels, while the average CV% of %IOD was 45.7% in the NEPHGE gels. Addition of interleukin-1beta (IL-1beta) to the cultures resulted in statistically significant modulation or de novo synthesis of 105 proteins in the 10% gels. In conclusion, we present the first 10% and 15% acrylamide 2-D gel protein databases of neonatal rat islets of Langerhans and demonstrate its usage to identify proteins altered in expression by IL-1beta.
胰岛素依赖型糖尿病是由朗格汉斯岛中β细胞的自身免疫性破坏引起的。细胞因子白细胞介素1抑制胰岛素释放，并且对分离的大鼠胰腺胰岛中的β细胞具有选择性细胞毒性。引发免疫反应的抗原以及白细胞介素1介导的β细胞细胞毒性的细胞内作用机制尚不清楚。然而，先前的研究发现β细胞破坏与蛋白质合成改变有关。因此，胰岛蛋白质的二维（2-D）凝胶电泳可能是促进胰岛素依赖型糖尿病分子发病机制研究的重要工具。胰岛蛋白质的二维凝胶电泳可能会导致：（i）确定细胞因子诱导的特定胰岛蛋白质的定性和定量变化；（ii）确定调节细胞因子作用的药物的效果；（iii）鉴定引发β细胞免疫破坏的主要胰岛蛋白质抗原。因此，本研究的目的是创建在标准化培养条件下标记的新生大鼠胰岛10%和15%丙烯酰胺二维凝胶上所有可重复检测到的蛋白质斑点的数据库（DB）（10%和15%DB）。在15%等电聚焦（IEF）和非平衡pH梯度电泳（NEPHGE）数据库中，分别在5块凝胶中的5块中出现了1235个和557个斑点，而在10%IEF和NEPHGE数据库中，分别在5块凝胶中的5块中出现了995个和378个斑点，斑点检测的重现性在75.2%至91.7%之间。在两个数据库中，所有凝胶中出现的斑点的积分光密度百分比的平均变异系数（%IOD的CV%）在42.4%至45.7%之间。当在不同日期的连续凝胶组中分析同一样品（批间分析）时，%IOD的平均CV%为35.5%-36.1%。当在一组凝胶中重复分析同一样品（批内分析）时，IEF凝胶中%IOD的平均CV%为30.2%，而NEPHGE凝胶中%IOD的平均CV%为45.7%。向培养物中添加白细胞介素-1β（IL-1β）导致10%凝胶中105种蛋白质发生统计学上显著的调节或从头合成。总之，我们展示了首个新生大鼠朗格汉斯岛胰岛10%和15%丙烯酰胺二维凝胶蛋白质数据库，并证明了其用于鉴定受IL-1β影响而表达发生改变的蛋白质 的用途。
