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研究生:陳縈欣
研究生(外文):Ying-HsinChen
論文名稱:核內呼吸因子和整合相關蛋白抑制人類髓母細胞瘤之增生
論文名稱(外文):Nuclear Respiratory Factor 1 and Integrin-Associated Protein inhibit the proliferation of human medulloblastoma cells
指導教授:黃阿敏黃阿敏引用關係
指導教授(外文):A-Min Huang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:55
中文關鍵詞:人類髓母細胞瘤核內呼吸因子整合相關蛋白
外文關鍵詞:medulloblastomanuclear respiratory factor 1integrin-associated protein
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腦部發育過程中,複製能力不受控制的前驅細胞會導致腦腫瘤的形成。髓母細胞瘤是一種常見於小腦的腫瘤,是發育中的小腦顆粒細胞過度複製所致。研究指出,在髓母細胞瘤中整合相關蛋白基因(Integrin-associated protein,IAP)的表現量明顯比正常小腦顆粒細胞少。然而,IAP在髓母細胞瘤中的功能仍然不清楚。我們實驗室發現,在小腦發育過程中,停止複製的顆粒細胞其IAP表現量明顯上升。另外也發現,調控IAP表現量的核內呼吸因子(Nuclear respiratory factor 1,NRF-1)可以促進神經母細胞瘤的分化。因此我們認為NRF-1和IAP可以抑制髓母細胞瘤的複製。本研究使用髓母細胞瘤Daoy細胞株進行研究。首先利用半定量反轉錄聚合酶連鎖反應確認在Daoy細胞株NRF-1和IAP均有表現。利用溴化去氧尿苷來檢測細胞複製的能力,發現當過度表現NRF-1或IAP均可抑制細胞複製,反之,當抑制NRF-1或IAP的表現均明顯促進Daoy細胞複製的能力。在探討NRF-1和IAP對細胞複製抑制作用的訊息機制中,也發現促分裂素原活化蛋白激酶(mitogen-activated protein kinases,MAP激酶,MAPK)上游的MAPK激酶(MEK)抑制劑U0126消除了NRF-1和IAP的抑制作用。此外,減少NRF-1和IAP會增加細胞週期蛋白D1的表現。實驗進一步發現抑制NRF-1以及IAP的表現增加了Daoy形成細胞群落的能力;反之,利用IAP的促效劑4N1K,減少了細胞群落的數量。在免疫缺陷鼠注射Daoy細胞1~2個月後,可觀察到腫瘤的形成,若注射降低IAP表現量的Daoy細胞,則顯著增加其腫瘤體積,而利用4N1K活化IAP則可以抑制腫瘤生長。以上發現指出,在髓母細胞瘤NRF-1和IAP會藉由促分裂素原活化蛋白激酶的訊息傳遞路徑以及細胞週期蛋白D1來調控細胞的複製,IAP表現量的減少促進腫瘤生長,此外活化IAP將會是一個具有潛力治療髓母細胞瘤的藥物。
During brain development, the uncontrolled proliferation of precursors results in tumorigenesis. Medulloblastoma is the most common malignant brain tumor in children and is thought to result from the highly proliferated granule cell precursors (GCPs) in the developing cerebellum. Studies have shown that integrin-associated protein (IAP) is one of the genes down-regulated in mouse medulloblastoma, as compared to GCPs. However, the role of IAP in medulloblastoma remains unknown. Our recent findings revealed that the expression of IAP is up-regulated in post-mitotic granule neurons in the developing mouse cerebellum. We also found that IAP is critically regulated by the transcription factor nuclear respiratory factor 1 (NRF-1) which promotes neuronal differentiation in human neuroblastoma cells. In this study, we hypothesized that up-regulation of NRF-1 or IAP inhibits cell proliferation in medulloblastoma. We used the human medulloblastoma Daoy cells for investigation. The expression of NRF-1 or IAP gene in Daoy cells was confirmed by RT-PCR analysis. Overexpression of NRF-1 or IAP by lipofectamine transfection decreased the percentage of BrdU-positive cells while knockdown of NRF-1 or IAP increased the percentage of BrdU-positive cells, suggesting that up-regulation of IAP and NRF-1 inhibits Daoy cell proliferation. The MAPK inhibitor U0126 reversed the inhibition of cell proliferation by overexpression of NRF-1 or IAP. In addition, when IAP was knocked down, the expression of cell cycle regulator cyclin D1 was up-regulated. Colony formation assays showed that knockdown of NRF-1 or IAP increased the number of colonies, while treatment with the IAP agonist peptide 4N1K significantly decreased the number of colonies in a dose dependent manner. Injection of Daoy cells into NOD-SCID mice, tumors were observed 1 to 2 months later. Tumor volume increased in NOD-SCID mice injected with Daoy cells containing shIAP while activation of IAP by 4N1K inhibits tumor growth in vivo. Our findings indicate that NRF-1 and IAP inhibit the proliferation of medulloblastoma cells via the MAPK pathway and cell cycle regulator cyclin D1. The down-regulation of the expression of IAP increases the tumor growth in vivo. Our results also suggest that activation of IAP is a potential therapeutic target for the treatment of medulloblastoma.
中文摘要 i
Abstract ii
誌謝 iv
Contents vi
Figure contents viii
Introduction 1
I. Medulloblastoma 1
i. Medulloblastoma formation 1
ii. Normal cerebellar development 1
II. Differential expression of genes between normal GCPs and medulloblastoma cells 2
III. Integrin-Associated Protein (IAP) 3
i. The structure of IAP 3
ii. The ligands of IAP 3
iii. The functions of IAP 4
IV. Nuclear Respiratory Factor-1 (NRF-1) 6
i. The structure of NRF-1 6
ii. Neural function of NRF-1 6
V. The relationship of NRF-1 and IAP 8
i. NRF-1 is a critical transcription factor of IAP 8
ii. NRF-1 promotes neurite outgrowth through the expression of IAP 8
VI. Research rationale 9
Materials and methods 10
I. Cell line and cell culture 10
II. Animals 10
III. RNA isolation and reverse transcription (RT)-PCR 11
IV. Transfection 12
V. Recombinant lentivirus production and infection 12
VI. Bromodeoxyuridine (BrdU) incorporation 13
VII. Colony formation analysis 13
VIII. Preparation of protein extracts 14
IX. Western blotting 14
X. Immunofluorescence microscopy 15
XI. Tumor growth in NOD-SCID mice 16
XII. Statistical analysis 17
Results 18
I. Expression of IAP and NRF-1 genes in human medulloblastoma Daoy cells 18
II. Up-regulation of IAP inhibits the proliferation of Daoy cells 18
III. Up-regulation of NRF-1 inhibits the proliferation of Daoy cells 19
IV. U0126 reverses the inhibition of cell proliferation by overexpression of NRF-1 and IAP in Daoy cells 20
V. Knockdown of IAP increases cyclin D1 expression 21
VI. Knockdown of NRF-1 and IAP increases the colony number of Daoy cells 22
VII. Knockdown of the expression of IAP but not NRF-1 increases tumor growth in vivo 22
VIII. Activation of IAP by 4N1K, an IAP agonist peptide, decreases colony formation in Daoy cells 23
IX. Activation of IAP by agonist peptide 4N1K decreases the tumor size in NOD-SCID mice 24
Discussion 25
I. Summary of this study 25
II. NRF-1 and IAP regulate the proliferation of human medulloblastoma cells 25
III. The signaling mechanisms of up-regulation of NRF-1 and IAP on the inhibition of medulloblastoma cell proliferation 27
IV. The IAP agonist 4N1K as a potential therapeutic drug for medulloblastoma 28
Conclusion 31
References 32

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