以二維電泳探討紅血球形成過程中蛋白基因體之表達變動

紅血球的生成對人體健康是一個重要的生物過程，它調控了用來運送呼吸作用所需氣體的紅血球。紅血球生成的病變或衰退將導致貧血至組織缺氧病變敗亡。有許多的體外細胞培養實驗可以利用液態或甲基纖維素培養基將造血幹原細胞誘導分化為紅血球。紅血球生成（ erythropoiesis）指的是造血幹原細胞分化到成熟紅血球的過程，但是調控紅血球生成的分子機制至今仍不明瞭。為了能進一步明瞭用來參與、催化、調控紅血球生成的細胞活性分子，本研究選擇以定位酸鹼梯度二維電泳（ immobilized pH gradients two-dimensional electrophoresis）來探討紅血球生成過程中，其蛋白基因體（ proteomic）表達變動的分析圖譜。首先我們應用正常人、 acute myeloid leukemia（ AML）病人及經體外增生具 burst forming unit-erythroid（ BFU-E）形成缺陷的 CD34+ cells（ expanded CD34+ cells day19）。二組二維電泳圖譜比較分析發現了 12個可能與喪失紅血球形成能力相關蛋白分子。以二維電泳圖譜比較的分析結果，相對於正常的 CD34+ cells發現 AML病人與增生 19天的 CD34+ cells兩者有 5種類的蛋白質減少表現。在這兩組具形成紅血球缺陷的細胞中並沒有共同新增加的蛋白質（相對於正常的 CD34+ cells）。減少的 5種細胞蛋白質分子量介於 11-19kDa。此外， CD34+ cells day19中發現了一個分子量約 38kDa（ pI 8.2）的蛋白分子表現量增加。在 AML病人的 CD34+ cells中則沒有明顯的蛋白分子表現量增加。了解紅血球生成過程中蛋白體表達變動，特別是 BFU-E、 CFU-E與未成熟紅血球階段之二維電泳分析目前正在進行中，成功的完成此部份的分析可望增進紅血球生成的細胞蛋白體變動相關機制之解明。

An in vitro Study of the Proteomic Expression in Human Red Blood Cell Formation by IPG 2D Electrophoresis

Red Blood Cell (RBC) formation, erythropoiesis, is a vital important bio-process to the human health. It regulates the appropriated supply of respiratory gases (O2 and CO2) to support the life of human body. Defects in the normal process of erythropoiesis may cause to various ischemic and anemic syndromes. Many in vitro models of erythropoiesis derived from hematopoietic progenitor cells, CD34+ cells, have been reported based on the liquid culture or semisolid medium colony-forming assay. However, the molecular mechanism that controls the normal process of erythropoiesis needs further elucidation. In order to study the proteins that specifically involved in catalyzing and controlling the erythropoietic differentiation, in this study we have characterized the dynamic changes of proteomic-expression profiles in the course of, using an immobilized pH gradients (IPG) two-dimensional electrophoretic-mapping (IPG-2DE) analysis. In this report we are presenting the preliminary results obtained from a comprehensive analysis of protein expression profiles in normal, abnormal and ex vivo expanded day 19 CD34+ cells (defect in BFU-E formation) by the 2DE. Approximately nine proteins have been identified in related to the lost of erythropoietic differentiation potentials of the normal CD34 cells. A comparative 2DE mapping analysis of the cell proteins obtained from an acute myeloid leukemia (AML) patient and 19 days expanded CD34+cells has led to identify the common loss in synthesis of five proteins in these erythropoietic defective progenitors, while no common proteins are identified with the increase synthesis in the two defective CD34 cells. These proteins with molecular weight ranged from 11-19KDa level were characterized. On the other hand, increased synthesis of one protein with molecular weight at level of 38KDa (pI 8.2) was identified in the expanded cells, but no apparent protein was characterized in the patient’s cells. Further characterization of these proteins and identification of protein expression profiles in the course of RBC formation, especially in BFU-E, CFU-E, immature and mature erythroblasts stages of erythropoiesis are undertaken.