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研究生： 許景然
Hsu, Jin-Ran
論文名稱： 胞外訊息調控激脢參與在維他命A酸誘導之人類神經分化之研究
Involvement of Extracellular Signal-Regulated Kinase in Retinoic Acid-Induced Human Neuronal Differentiation
指導教授： 陳淑姿
Chen, Shur-Tzu
黃浩仁
Huang, Hao-Jen
學位類別： 碩士
Master
系所名稱： 醫學院 - 細胞生物與解剖學研究所
Institute of Cell Biology and Anatomy
論文出版年： 2003
畢業學年度： 91
語文別： 英文
論文頁數： 40
中文關鍵詞： 維他命A酸 、神經分化
外文關鍵詞： N-cadherin, neuronal differentiation
相關次數： 點閱：153  下載：1 
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    • 哺乳類動物中樞神經系統中的神經分化及發育是經由一連串事件發生累積而來的。 而MAP (mitogent-activated protein) kinase已被證實在神經元分化及凋亡的過程中扮演一個重要的角色。在前人的實驗中已證實一衍生自人類畸胎瘤的細胞株hNT2 (NT2) cell是一個用來研究神經分化很好的模型；經由維他命A酸 (RA) 處理NT2細胞數週之後，細胞會分化成不再具分裂能力的神經細胞並且表現出成熟神經細胞的標定物質。在我的研究發現在處理RA的過程中，ERK1/2磷酸化的程度隨著時間而升高，並且在RA處理的第七天細胞有聚集的現象。以RA及MEK抑制劑U0126同時處理NT2細胞發現ERK1/2的磷酸化程度下降而且細胞聚集的情形也被破壞，但是細胞在型態上以及neurofilament middle-chain (NF-M)的表現上並沒有差異，這暗示說ERK1/2參與在RA所誘發的神經分化，而且與細胞的聚集有關。進一步的研究發現，N-cadherin這個細胞附著分子的表現與ERK1/2的活化程度表現一致。這些結果顯示出磷酸化的ERK1/2可以藉由調控N-cadherin的表現來影響神經細胞的聚集。此研究結果可為神經分化及其生理功能上提供一些新的思維。

    Neuronal differentiation in the mammalian CNS is driven by multiple events. Mitogen activated protein kinases (MAPKs) have been observed to play roles in neuronal development. It have been demonstrated that hNT-2 cells (NT2 cells), a cell-line derived from human teratocarcinoma, act as be a good model for study neuronal development. Following retinoic acid (RA) treatment, NT-2 cells can differentiate into postmitotic neuronal cells and express several mature neuronal markers. In this thesis, during period of RA induction, phosphorylated form of extracellular signal-regulated protein kinases1/2 (ERK1/2; members of MAPKs) raised and cell aggregation was also observed at the 7th day after RA induction. In addition, co-treatment with RA and MEK1/2 inhibitor, U0126, phospho-ERK1/2 were down-regulated and cell aggregation was also abrogated, while no detectable change in cellular morphology and the expression of neurofilament middle-chain (NF-M) were observed. It implies that ERK1/2 may take participated in the process of RA-induced neuronal differentiation and in charge for cellular aggregation during neuronal development. Furthermore, the expression of N-cadherin, a cell adhesion molecule, is consistent with the alteration of phosphor-ERK1/2 level. These data indicate that ERK1/2 regulate the N-cadherin molecule expression to modulate cell aggregation. The results may provide a thought for further explore the physiological functions in the process of neuronal differentiation.

    ACKNOWLEDGEMENTS I CHINESE ABSTRACT II ABSTRACT III CONTENTS IV LIST OF TABLES VI LIST OF FIGURES VII ABBREVIATION VIII I. INTRODUCTION 1 1. Neuronal differentiation of the central nervous system 1 2. hNTera2/c. D1 cell line 2 3. MAP kinase 3 3.1 Discovery of MAP kinase 4 3.2 MAP kinase pathways 5 3.3 ERK MAP kinase pathway 6 3.4 p38 MAP kinase pathway 7 3.5 JNK MAP kinase pathway 8 4. MAP kinase and neuronal differentiation 9 5. Aims of this study 11 II. MATERIALS AND METHODS 12 1. Cell Culture 12 2. Antibody 12 3. Preparation of Protein Extracts 13 4. Electrophoresis and Western Blot Analysis 13 5. Immunofluorescent Staining 14 III. RESULTS 15 1. RA induce NT2 cell differentiation 15 2. RA activates ERK1/2 phosphorylation and NF-M expression in a time-dependent manner 15 3. Inhibition of ERK1/2 activation by MEK inhibitors 16 4. RA-induced cell aggregation of NT2 cells is decreased after inhibition of ERK1/2 activation 16 5. N-cadherin might be the downstream target of ERK1/2 activation 17 IV. DISCUSSION 18 V. REFERENCES 22 LIST OF TABLES Table 1. The origins, advantages and inductive agents of three different neuronal cell lines 31 Table 2. The different MAP kinase pathway and their relative functions in cells 31 LIST OF FIGURES Figure 1. Morphologic appearance of NT2 cells in feeding medium at different time course 32 Figure 2. NT2 cells differentiated under 10μM RA induction at different time course 33 Figure 3. Immunofluorescence staining of RA-induced NT2 cells 34 Figure 4. Western blotting and statistical analysis of cell lysate of normal culture cell and RA-inducing cell 35 Figure 5. The effects of RA on NF-M protein expression in NT-2 cells 36 Figure 6. Mophologic observationof NT2 cells cotreat with RA and MEK inhibitor, U0126 in different time course 37 Figure 7. ERK, phospho-ERK and NF-M expression between cells in different treatments and time course 38 Figure 8. The effects of RA and U0126 on ERK activation of NT2 cells 39 Figure 9. Western blot analysis of N-CAM and N-cadherin expression 40

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