臺灣博碩士論文加值系統

巨核細胞(megakaryocyte)是血小板的前驅細胞，當megakaryocyte成熟之後會由細胞質裂解，形成血小板釋放到血液中，執行血液凝集的功能。在megakaryocyte及血小板的細胞膜上會表現大量的integrin (alpha)IIb(beta)3並且透過內攝作用(endocytosis)將fibrinogen從血漿中攝入並儲存在(alpha)-granule中。在megakaryocyte往血小板分化的過程中，一個已知與receptor endocytosis相關的蛋白Disabled-2 (DAB2)的表現量會增加，同時在血小板活化後DAB2會釋放並與integrin (alpha)IIb(beta)3產生交互作用，影響血小板與fibrinogen的結合以及aggregation的能力。透過以上文獻的支持，我的研究目的在了解megakaryocyte往platelet分化的過程中，DAB2是否也會參與integrin (alpha)IIb(beta)3將fibrinogen吞入細胞的過程，除此之外也想要了解DAB2會透過何種方式影響integrin (alpha)IIb(beta)3對fibrinogen進行的endocytosis。首先利用K562細胞株處理TPA建立的血球分化系統，往megakaryocyte分化的細胞具有內噬 fibrinogen的能力，同時發現在DAB2-knockdown的細胞，fibrinogen內吞的能力會下降，表示在megakaryocyte-like cell進行內攝作用的過程中的確有DAB2的參與。另一方面建立了細胞轉染系統，在CHO細胞上表現integrin (alpha)IIb(beta)3觀察對fibrinogen內攝作用的影響，已知表現活化態的integrin (alpha)IIb(beta)3具有內噬 fibrinogen的能力，代表fibrinogen的endocytosis是(alpha)IIb(beta)3 dependent pathway。進一步利用免疫螢光染色的方式觀察fibrinogen、DAB2以及clathrin在細胞內的分佈，初步發現此三種蛋白之間有交互作用，同時fibrinogen的內攝作用會被hypertonic buffer所抑制，此結果證明 megakaryocyte內噬fibrinogen主要是透過clathrin¬dependent endocytosis pathway。此篇研究首次證明DAB2會與clathrin complex產生交互作用，並且調控megakaryocyte在分化過程中內噬fibrinogen的能力。

Megakaryocytes are derived from hematopoietic stem cell in the bone marrow, and express integrin (alpha)IIb(beta)3 that can bind and uptake their ligand fibrinogen and stored in (alpha)-granule during megakaryocyte maturation. An endocytosis associated protein Disabled-2 (DAB2) is up-regulated and translocates to membrane interacting with integrin (beta)3 in the progress of megakaryocyte differentiation. Based on these studies, we are interested in investigating whether DAB2 is involved in integrin (alpha)IIb(beta)3-mediated fibrinogen endocytosis, and in integrin (alpha)IIb(beta)3 signaling pathway. First we used K562 cell line to establish the hematopoietic cell differentiation system. We found that megakaryocyte-like cells can uptake fibrinogen but this endocytosis was decreased in DAB2-knockdown cells. In addition, we used(alpha)IIb(beta)3-transfected CHO-K1 cells as a model system to confirm that fibrinogen endocytosis is an integrin (alpha)IIb(beta)3 dependent mechanism. Furthermore, we utilized the immunofluorescence staining to observe the distribution of fibrinogen, DAB2 and clathrin, and found they colocalized with each other. We also found that fibrinogen endocytosis can be blocked with hypertonic buffer, it means endocytosis of fibrinogen by megakaryocytes occurs via a clathrin-dependent process. This study demonstrates for the first time that DAB2 interacts with clathrin complex that play a role in the regulation of fibrinogen endocytosis during megakaryocytic differentiation.

指導教授推薦書............................................
口試委員會審定書..........................................
授權書...................................................iii
致謝.....................................................iv
中文摘要.................................................v
英文摘要.................................................vi
縮寫表...................................................vii
第一章、Background and Significance......................1
1.DAB2發現與結構.........................................1
2.DAB2功能..............................................2
3.DAB2與血球分化.........................................3
4.DAB2與endocytosis.....................................3
5.Integrin..............................................4
6.Fibrinogen............................................5
7.Specific Aims.........................................6
第二章、Materials and Methods............................8
1.實驗材料...............................................8
2.實驗方法...............................................10
第三章、Result...........................................18
第四章、Discussion.......................................31
第五章、References.......................................39
圖表.....................................................45
圖1、建立血球分化系統......................................45
圖2、利用螢光顯微鏡觀察K562 cell endocytosis的現象..........46
圖3、利用flow cytometry偵測K562 cell endocytosis的能力.....47
圖4、觀察DAB2-difficient對endocytosis的影響................48
圖5、偵測K562-V7、si8 cell integrin (alpha)IIb(beta)3表現..50
圖6、觀察siDAB2表現對endocytosis的影響......................51
圖7、利用共軛焦顯微鏡觀察siDAB2表現對endocytosis的差異........53
圖8、觀察rescue integrin (alpha)IIb(beta)3表現對endocytosis
的影響....................................................54
圖9、建立CHO-K1 轉染細胞...................................55
圖10、偵測integrin (alpha)IIb(beta)3 endocytosis的能力.....56
圖11、偵測K562-V7、Si8細胞中clathrin、caveolin表現..........58
圖12、觀察K562-V7、si8T cell中clathrin、AP-2分佈與表現......59
圖13、觀察K562-V7、si8 cell中clathrin、DAB2分佈與表現.......60
圖14、觀察K562-V7、si8T cell中clathrin、fibrinogen分佈與表現.61
圖15、觀察hypertonic buffer對K562-V7、si8 cell endocytosis
的影響....................................................62
圖16、觀察K562-V7、si8 cell中DAB2、fibrinogen分佈與表現.....63

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