2015-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/659207摘要：紅血球生成是一個嚴密調控的步驟，需要由生長分裂中的紅血球前驅細胞分化成成熟的紅血球。目前所熟知會調控紅血球生成的因子有紅血球生成素(EPO)、GM-CSF 及TGF-。EPO 會促進紅血球前驅細胞分裂、存活及分化。GM-CSF 會促進紅血球前驅細胞分裂及存活。TGF-會抑制紅血球前驅細胞分裂但會促進分化。雖然EPO 及GM-CSF 已知是經由活化受體誘導JAK2- STAT5 下游訊息傳遞，我們近期研究發現非受器型酪胺酸激酶Syk 也參與EPO 及GM-CSF 的訊息傳遞。Galectin-1 (GAL1)是β-半乳糖甙結合動物凝集素(galectin)的成員之一，在發育過程中扮演重要角色。儘管GAL1 不具有訊息序列 (signal peptide)，細胞質內的GAL1 仍可藉非正常的釋放程序被分泌到胞外。GAL1的過度表達已知與癌細胞的癌化有關，然而相較於GAL1 功能在癌症生物學領域的廣泛性了解，其在紅血球生成的病生理角色尚未明確。依據現有少數的文獻報導，GAL1 蛋白的表現量會伴隨紅血球血癌細胞株TF-1 分化時減少。此外紅血球獲得其成熟的的表現型與GAL1 分泌到胞外並結合至細胞表面有關，且GAL1 結合至細胞表面的現象可被半乳糖甙所抑制。這些發現暗示GAL1 可能是一有效調控紅血球分化的分子，且此作用可能是經由細胞外的作用路徑而來。在我們初步的研究中發現抑制GAL1 時會降低EPO及GM-CSF 的細胞存活作用，而EPO 會抑制GAL1 的mRNA 表現但却不影響細胞中的蛋白質量。在這個計劃中，我們將利用紅血球血癌細胞株TF-1 及K562，與GAL-1 剔除的小鼠初代紅血球前驅細胞，了解GAL1 在紅血球生成的細胞功能及分子機轉。目標一： 探討GAL1 影響EPO、GM-CSF 及TGF-β 調控紅血球前驅細胞存活、生長及分化之角色。目標二：探討GAL1 調控紅血球前驅細胞存活、生長及分化的分子機轉。目標三：探討GAL1 以自泌素作用方式影響紅血球生成的機制。目標四：探討GAL1 基因及蛋白表現的調節分子機制<br> Abstract: Erythropoiesis is a tightly regulated process of differentiation from erythroid progenitorcells to mature erythrocytes. Several growth factors are involved in erythropoiesis, and theprincipal factors regulating this process include erythropoietin (EPO), granulocytemacrophage colony-stimulating factor (GM-CSF) and transforming growth factor- (TGF-).EPO can support proliferation, survival and differentiation of erythrocyte progenitor cells,GM-CSF can support proliferation and survival, TGF- inhibits cell proliferation butpromotes cell differentiation. Although JAK2/STAT5 signaling is recognized the majorinitiator of EPO and GM-CSF receptors signaling, our recent study indicates that spleentyrosine kinase (Syk) is also the downstream signaling molecule for both receptors inerythrocyte progenitor cells.Galectin-1 (GAL1) is a member of the family of beta-galactoside binding animal lectins,and its expression is developmentally regulated. Although devoid of signal peptide cytosolicGAL1 can be externalized from cells by a mechanism independent of the normal secretoryprocess. Compared to the wide and extensive understanding on the function of GAL1 incancer biology, i.e. GAL1 overexpression is associated with neoplastic transformation andloss of differentiation, its physiological and pathological roles in erythropoiesis remain largelyunknown. According to the limited studies, a decreased GAL1 protein expression wasobserved in TF-1 cells along with differentiation. Moreover, acquisition of an erythroidphenotype is associated with an externalization of GAL1 and it can bind to cell surface in agalactoside-inhibitable fashion, suggesting that GAL1 might be an effective molecule toregulate erythrocyte differentiation and functions through extrinsic pathway. Our preliminarydata reveal that GAL1 inhibition or silencing can reduce cell survival induced by EPO andGM-CSF, and EPO can inhibit GAL1 mRNA expression but does not affect cellular GAL1protein level. In this study, we will use TF-1 and K562 erythroleukemic cell lines as well asgalectin-1 knockout erythrocyte progenitor cells to understand the cellular function andmolecular action mechanisms of GAL1 in erythropoiesis.Aim 1: To understand the coordinative role of GAL1 with EPO, GM-CSF and TGF- inregulating survival, proliferation and differentiation of erythroid progenitor cells.Aim 2: To understand the molecular mechanisms of GAL1 in regulating survival,proliferation and differentiation of erythroid progenitor cells.Aim 3: To understand the molecular mechanisms for the autocrine effect of GAL1.Aim 4: To understand the regulation mechanisms for GAL1 gene and proteinexpression.