細胞激素調控細胞增殖及分化訊息傳遞路徑 之研究

細胞激素調控細胞增殖及分化訊息傳遞路徑 之研究

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Part I:

Activin A 對於細胞的分化及細胞凋亡扮演重要角色。其中包括骨髓中的紅血球分化。 Basic fibrobl ast growth factor(bFGF) 是由 bone marrow stromal cell 所產生，它在造血幹細胞中，可以維持 primiti ve erythroid cell 的增生。然而， activin A 及 bFGF 以何種機制來使細胞分化或增生並不是很清楚。

本篇論文中我們探討了 bFGF 可以抑制 activin A 誘導紅血球的分化。此外，此篇論文也探討了 activ in A 及 bFGF 調控 K562 細胞分化時，不同的 mitogen-activated protein kinase 所扮演的角色。在西方 墨點法分析中顯示出：當細胞加入 activin A 後， p38 的磷酸化程度增加，而 MAPK 及 JNK 則被抑 制。另外，當細胞加入 bFGF 後，三種 MAPK 蛋白質磷酸化都被抑制。細胞若同時加入 activin A 及 bFGF ，則 p38 磷酸化被抑制。此外， activin A 誘導紅血球分化過程中，若以 U0126 抑制 MAP K 的活性，發現紅血球分化程度增加，若以 SB203580 抑制 p38 的活性，則會抑制 hemoglobin 的產 生。綜合以上結果得知， activin A 正調控 p38 磷酸化，而 bFGF 負調控 p38 磷酸化使紅血球分化。

Part : Ⅱ

細胞激素可以調節 hematopoietic cells 的生長、增生、分化及抑制細胞凋亡。當細胞激素將訊息由

細胞膜傳入到細胞核後，會改變基因，最後影響細胞的活性。在細胞激素調控 hematopoiesis 時， J

anus kinase/signal transducer and activator of transcription (JAK/STAT) pathway 扮演重要角色。大部份

的細胞激素可以活化兩個或兩個以上的 JAKs 。當 IL-3/IL-5/GM-CSF 和其接受子結合後會活化 JAK

1 及 JAK2 。 IL-3 、 IL-5 及 GM-CSF 接受子各自含有自己的 α 次單元，並共用 β 次單元。然而， J

AKs 以哪一部位和 IL-3 接受子結合並不清楚。先前實驗室已將 IL-3 接受子 α 次單元的 intracellular

cDNA 及 βc 的 intracellular cDNA 接到 GST 形成融合基因，此融合基因可以產生融合蛋白。本篇論

文中已得知 IL-3Rα 次單元可以和 JAK2 結合而 IL-3Rβc 次單元可以和 JAK1 結合。此結果和 IL-5 sy

stem 相同 (Blood 91:2264-71, 1998) ，因此，我建構一系列去除某區域的 JAK1 蛋白質和 JAK2 蛋白

質。接下來將以 GST-pull down assay 來探討 JAK1 和 JAK2 以哪一部位和 IL-3 接受子結合，並且研

究此區域對 IL-3 接受子訊號傳遞及 IL-3 依賴型細胞生長活性的影響。

Signal transduction pathways involved in the regulation of cell proliferation and differentiat

ion by cytokines

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Part I:

Activin A is essential for cell differentiation and apoptosis including erythroid cell differentiation in the bone marrow. Basic fi broblast growth factor (bFGF) is produced by bone marrow stromal cells and by hematopoietic cells that can maintain prolifer ation of primitive erythroid cells. However, the mechanism by which activin A and bFGF exert their differentiation or prolifer ation activity is still unknown. In this study, I have investigated the bFGF inhibit the effect of activin A-induced erythroid diff erentiation. Furthermore, this study examined the role of different mitogen-activated protein kinase signal transduction pathw ays in activin A and bFGF-modulated differentiation of K562 cells. Western blot analysis of a panel of phosphorylated protein s revealed that the phosphorylation of p38 is increased and the phosphorylation of MAPK and JNK are inhibited after the cells of treatment with activin A. In addition, the phosphorylation of three MAPKs are inhibited after the cells of treatment with bF GF. The phosphorylation of p38 is inhibited after the cells of treatment with activin A and bFGF. Moreover, inhibition of MA PK activity by U0126 induced erythroid differentiation, whereas inhibition of p38 activity by SB203580 inhibited induction o f hemoglobin production by activin A. These results suggest that the phosphorylation of p38 is positively regulated by activin A and is negatively regulated by bFGF in erythroid differentiation.

Part : Ⅱ

Cytokines regulate the growth, proliferation, differentiation and apoptosis prevention of hematopoietic cells. Cytokine signali

ng from cell membrane into the nucleus in which the genes are altered, and eventually cell activity are affected. Janus kinase/s

ignal transducer and activator of transcription (JAK/STAT) pathway play a very important role among the cytokine-regulated

hematopoiesis. Most cytokines can activate two or more than two JAKs. When IL-3/IL-5/GM-CSF bind to their receptors, init

iating activation of JAK1 and JAK2. The receptors for IL-3, IL-5 and GM-CSF each consist of a cytokine specific a chain, an

d a common b-chain (bc). However, which regions of JAKs interact with IL-3 receptor are now unclear. In previous study, the

intracellular cDNA of IL-3 receptor a subunit and the intracellular cDNA of bc were fused with GST form fusion genes. Thes

e fusion genes can produce fusion proteins. In this study, the data show that IL-3R a chain interacts with JAK2 and IL-3R bc i

nteracts with JAK1. These results consistent with the published data for IL-5 system (Blood 91: 2264-2271, 1998). Hence, I c

onstructed a series of deletion mutants for JAK1 and JAK2. In the future, the regions of JAK1 and JAK2 interact with recepto

rs by GST-pull down assay and the effect of these regions on IL-3 receptor signal transduction as well as survival activity in I

L-3-dependent cells will be investigated.