ADDUCIN ISOFORMS IN RBC AND PLATELET DIFFERENTIATION

红细胞和血小板分化中的内收肌蛋白异构体

• 批准号: 6799135
• 负责人: Diana Gilligan
• 金额: $ 6.5万
• 依托单位: BLOODWORKS
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6799135
• 关键词: actins; cell differentiation; crosslink; erythrocytes; fluorescence microscopy; gene expression; gene targeting; genetic promoter element; genetically modified animals; human tissue; immunofluorescence technique; laboratory mouse; megakaryocytes; membrane proteins; messenger RNA; phosphorylation; platelet activation; platelet aggregation; platelets; polymerase chain reaction; protein isoforms; protein kinase C; protein structure function; spectrin; western blottings

The goal of this research is to test the hypothesis that the membrane skeleton protein adducin is critical to the assembly of the membrane skeleton during differentiation of erythrocytes and platelets. The membrane skeleton is crucial to the red cell, providing both support and flexibility as cells move rapidly through the circulation and traverse narrow capillaries. Defects in membrane skeleton proteins cause mild to severe hemolytic anemia and even hydrops fetalis. Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases. The membrane skeleton is also crucial to normal platelet function. The following specific aims are designed to analyze adducin's role in both erythrocyte and platelet differentiation and function: I. Determine the role of alternatively spliced adducins in erythrocyte differentiation a. complete the analysis of adducin expression patterns during normal human and mouse erythroid differentiation. b. elucidate the role of adducin in erythroid differentiation and function in vivo using two approaches: adducin null "knockout" mice and transgenic mice in which an erythroid specific promoter directs antisense mRNA production to block adducin expression. c. perform molecular dissection of the functions of the alternatively spliced isoforms in vivo by using an erythroid specific promoter to direct overexpression of tagged adducin isoforms (normal or mutant) to compete with normal adducin function in transgenic mice or to rescue knockout mice. II. Determine adducin's role in megakaryocyte differentiation and platelet function a. determine adducin expression patterns in mature platelets versus megakaryocytes b. determine the role of adducin in activation and aggregation of platelets c. elucidate the role of adducin in megakaryocyte differentiation and platelet function in vivo using two approaches: adducin null "knockout" mice and transgenic mice in which a platelet specific promoter directs antisense mRNA production to block adducin expression. d. perform molecular dissection of the functions of the alternatively spliced isoforms in vivo by using a platelet specific promoter to direct overexpression of tagged adducin isoforms (normal or mutant) to compete with normal adducin function in transgenic mice or to rescue knockout mice.

本研究的目的是检验以下假设：膜骨架蛋白内收蛋白对于红细胞和血小板分化过程中膜骨架的组装至关重要。 膜骨架对红细胞至关重要，当细胞快速穿过循环系统并穿过狭窄的毛细血管时，膜骨架提供支撑和灵活性。 膜骨架蛋白缺陷会导致轻度至重度溶血性贫血，甚至胎儿水肿。 遗传性溶血性贫血（球形红细胞增多症或椭圆形红细胞增多症）是最常见的遗传性疾病之一。 膜骨架对于血小板的正常功能也至关重要。 以下具体目的旨在分析内收蛋白在红细胞和血小板分化和功能中的作用： I.确定可变剪接内收蛋白在红细胞分化中的作用。完成正常人和小鼠红系分化过程中内收蛋白表达模式的分析。 b.使用两种方法阐明内收蛋白在体内红细胞分化和功能中的作用：内收蛋白无效“敲除”小鼠和转基因小鼠，其中红细胞特异性启动子指导反义mRNA产生以阻断内收蛋白表达。 c.通过使用红细胞特异性启动子指导标记的内收蛋白同种型（正常或突变）的过度表达，以与转基因小鼠中的正常内收蛋白​​功能竞争或拯救基因敲除小鼠，对体内可变剪接同种型的功能进行分子解剖。 二.确定内收蛋白在巨核细胞分化和血小板功能中的作用确定成熟血小板与巨核细胞中内收蛋白的表达模式 b．确定内收蛋白在血小板激活和聚集中的作用 c．使用两种方法阐明内收蛋白在体内巨核细胞分化和血小板功能中的作用：内收蛋白无效“敲除”小鼠和转基因小鼠，其中血小板特异性启动子指导反义mRNA产生以阻断内收蛋白表达。 d.通过使用血小板特异性启动子指导标记的内收蛋白同种型（正常或突变）的过度表达，以与转基因小鼠中的正常内收蛋白​​功能竞争或拯救基因敲除小鼠，对体内可变剪接同种型的功能进行分子解剖。