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研究生:楊政道
研究生(外文):Cheng-Tao Yang
論文名稱:探討轉錄因子HOXB4促進人類間葉幹細胞體外增殖的機制
論文名稱(外文):Elucidation of the mechanism underlying the proliferation-promoting effect of HOXB4 on human mesenchymal stem cells
指導教授:蘇瑀蘇瑀引用關係
指導教授(外文):Yeu Su
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生物藥學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:54
中文關鍵詞:人類間葉幹細胞增殖機制
外文關鍵詞:HOXB4human mesenchymal stem cellsproliferationmechanism
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  由骨髓基質分離出來的成人間葉幹細胞(human mesenchymal stem cells;hMSCs)除了能分化成硬骨、軟骨和脂肪等間葉組織外,亦能分化成內胚層的肝細胞和外胚層的神經細胞,因此被認為是細胞或基因療法的極佳材料。但該細胞在體外的增殖與分化能力會隨著培養代數的增加而降低,而本研究的主要目的是要探討HOXB4促進間葉幹細胞體外增殖的作用機制。除了利用實驗室先前製作出的腺病毒AdHOXB4外,也製備了腺病毒AdcMyc。經MTS分析,發現HOXB4和c-Myc皆能促進成人間葉幹細胞的增殖,且在感染等量病毒的狀況下,HOXB4的效果優於c-Myc;另經錐蟲藍染色計數後,證實HOXB4具促進成人間葉幹細胞增殖的活性;而免疫螢光染色的結果,也顯示受AdHOXB4或AdcMyc感染的間葉幹細胞之增殖標記Ki67的訊號明顯高於對照組。反轉錄-聚合酶連鎖反應的分析結果亦指出在HOXB4和cMyc的高度表現間葉幹細胞中,c-Myc、cyclin D1、cyclin D3和E2F1表現增加,p21和p27表現降低;西方墨點法分析結果中顯示HOXB4和cMyc的高度表現間葉幹細胞中,c-Myc、c-Jun和p38表現增加,p21表現降低。由於HOXB4對c-Myc的影響甚微,推測其促進成人間葉幹細胞增殖應有c-Myc之外的途徑參與。此外,我們也利用酵母菌雙雜合系統來尋找能與HOXB4交互作用的分子,目前找到RanBPM、PIAS2�悕M一未知蛋白。綜言之,本研究的成果不但有助於我們對HOXB4之促進成人間葉幹細胞增殖機制的了解,也對未來研究的方向提供了良好的指引。
Human mesenchymal stem cells (hMSCs) derived from bone marrow can differentiate into cells of mesodermal (osteocytes, chondrocytes and adipocytes), endodermal (hepatocytes) and ectodermal (neurons) lineages. The multipotency of these cells makes them attractive materials for cell as well as gene therapies. However, the proliferation of hMSCs decreases during in vitro passage. Our previous work has shown that the homeodomain-containing transcription factor HOXB4 can enhance the in vitro growth of hMSCs. The aim of present study is to elucidate its underlying mechanism(s). Since c-Myc has recently been reported as a downstream effector of HOXB4 in promoting the self-renewal of hematopoietic stem cells (HSCs), recombinant adenoviruses were prepared and used to introduce HOXB4 and c-Myc genes, respectively, into hMSCs and their proliferation-enhancing effects were subsequently analyzed. Indeed, both HOXB4 and c-Myc significantly promote the proliferation of hMSCs based on MTS assay and cell counting, which was further supported by the results of Ki-67 immunofluorescence staining. RT-PCR and Western blotting were then used to assess the expression of several critical cell cycle regulators. While upregulation of cyclin D1, cyclin D3, E2F1, c-Jun, and p38 was found in both the HOXB4- and c-Myc-transduced hMSCs, expression of the CDK inhibitors p21 and p27 was downregulated in these cells. However, c-Myc is unlikely to be the main mediator of HOXB4 in stimulating hMSCs proliferation because expression of the former was only slightly increased by the latter. To identify the HOXB4-interating protein(s) that might alter its target recognition and binding affinity as well as its transactivating potential in hMSCs, a yeast two-hybrid screening was performed and three candidates including RanBPM, PIAS2��, and a novel one were identified. Together, our results provide not only the explanations for the role of HOXB4-induced proliferation of hMSCs but also basis for further direction of this study.
目錄……………………………………………………………………………………1
圖次目錄……………………………………………………………………………....2
英文摘要……………………………………………………………………………....3
中文摘要……………………………………………………………………………....4
緒論……………………………………………………………………………............5
實驗材料………………………………………………………………………………9
實驗方法……………………………………………………………………………..15
實驗結果…………………………………………………………………………......25
討論…………………………………………………………………………..............30
參考文獻……………………………………………………………………..……....34
圖表……………………………………………………………………………..........38
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