探討化合物Y抑制神經母細胞瘤轉移之效果

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蔡俊發(CHUN-FA TSAI) 查詢紙本館藏

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生命科學系

論文名稱

探討化合物Y抑制神經母細胞瘤轉移之效果
(Investigation on the metastasis inhibiting effects of compound Y in neuroblastom)

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摘要(中)

神經母細胞瘤（神母）是一種周圍神經系統的兒童癌症，約 50% 的神經母細胞瘤患者在診斷時已發生惡性轉移，因此開發抗轉移藥物是當前神母治療的當務之急。芳香烴受體（Aryl hydrocarbon receptor, AHR）是新發現的神母有利預後因子，AHR的表達與神母腫瘤的組織分化程度呈正相關，並預測患者更好的存活率。此外，我們之前的研究表明，內源性配體犬尿氨酸（Kynurenine, Kyn）激活AHR可顯著抑制神母轉移。然而，Kyn 限制神母轉移的有效劑量較高，這可能限制其作為治療藥物之潛力。化合物Y是我們新鑑定的AHR內源性配體，比Kyn具有更高激活AHR訊息傳遞路徑的能力，為了研究化合物Y是否也對神母轉移具有抑製作用，此研究利用了SK-N-BE(2)C 和 SK-N-SH 兩株神經母細胞瘤細胞來驗證化合物Y對轉移相關細胞行為的影響，包括細胞貼附、遷移和侵襲。首先透過化合物Y處理後的AHR下游基因CYP1A1增加證實化合物 Y 具活化AHR的效果。接著利用化合物 Y處理過後之細胞分別進行傷口癒合實驗、Tans-well 細胞侵犯實驗以及細胞貼附實驗，發現在給予化合物 Y治療過後之細胞的爬行能力與侵犯能力均有明顯下降的趨勢，相反地，細胞的貼附能力反而因為化合物 Y處理而增加，這些變化均與先前Kyn處理後之結果類似。此外，由於上皮細胞間質轉化（epithelial-mesenchymal transition，EMT）是癌症轉移的關鍵機制，為了解化合物 Y造成上述細胞行為變化之可能機制，我透過qPCR的方式觀察ＥＭＴ相關基因的變化，發現在化合物 Y處理過後會導致Vimentin、Slug的表達下降以及E-鈣粘蛋白（E-cadherin）的表達上調；同時也發現腫瘤轉移抑制基因KISS-1的mRNA表達也受到化合物 Y的刺激而上升，顯示其抑制轉移的潛力。總結，這項研究證實化合物Ｙ確實可以透過活化AHR進而調節KISS-1及ＥＭＴ相關基因的表達，從而抑制神經母細胞瘤細胞的轉移相關特性。

摘要(英)

Neuroblastoma (NB) is a childhood cancer of the peripheral nervous system. About 50% of patients with neuroblastoma have developed malignant metastases at diagnosis. Developing an anti-metastasis drug is urgent for the current NB treatment. Aryl hydrocarbon receptor (AHR) is a new-identified favorable prognostic factor of NB. The expression of AHR positively correlated with the differentiation histology of the NB tumors and predicted better survival of the patients. In addition, our previous study has suggested that activation of AHR by the endogenous ligand kynurenine (Kyn) significantly inhibits NB metastasis. However, the effective dose of Kyn for restricting NB metastasis is high which may limit its therapeutic potential. Compound Y is a novel endogenous ligand of AHR and shows a higher ability than Kyn to activate AHR signaling. To investigate whether compound Y also has inhibiting effects on NB metastasis, SK-N-BE(2)C and SK-N-SH NB cells were treated with compound Y followed by several in vitro analyses. First, the effect of compound Y on AHR activation was confirmed by the upregulation of CYP1A1. Then, to test compound Y’s effect on NB metastasis, wound healing, trans-well invasion assays, and cell adhesion assays were investigated. I found that compound Y inhibits the migration and invasion ability of the two NB cell lines. Reversely, compound Y treatments promote the adhesion of NB cells. These findings are similar to our previous observation of using Kyn as a treatment. Since epithelial-mesenchymal transition (EMT) is a key process of tumor metastasis, the EMT-related genes were analyzed by real-time PCR to verify the underlying mechanism of compound Y treatment. The results show that Vimentin and Slug were downregulated and E-cadherin was upregulated by compound Y. In addition, the tumor metastasis suppressor gene KISS-1 was also upregulated by compound Y indicating its anti-metastasis potential. Altogether, this study suggests that compound Y could affect the expression of EMT-related genes and KISS-1 through AHR activation, resulting in the restriction of NB metastasis.

關鍵字(中)

★ 神經母細胞瘤
★ 芳香烴受體

關鍵字(英)

★ Neuroblastoma
★ Aryl hydrocarbon receptor
★ AHR

論文目次

目錄
頁次
摘要----------------------------------------------------------------------------------------------------------I
Abstract---------------------------------------------------------------------------------------------------II致謝-------------------------------------------------------------------------------------------------------III
目錄---------------------------------------------------------------------------------------------------------V
第一章、緒論
1.1 神經母細胞瘤------------------------------------------------------------------------------- 01
1.1.1 介紹--------------------------------------------------------------------------------------01
1.1.2 神經母細胞瘤所導致的症狀與徵象-----------------------------------------------01
1.1.3 神經母細胞瘤癌症的分期-----------------------------------------------------------02
1.1.4 神經母細胞瘤的預後與治療方法--------------------------------------------------03
1.1.5 神經母細胞瘤的轉移因素-----------------------------------------------------------04
1.1.6 神經母細胞瘤遺傳標記 MYCN ---------------------------------------------------05
1.2 芳香烴受體 (AHR)------------------------------------------------------------------------07
1.2.1 介紹--------------------------------------------------------------------------------------07
1.2.2 AHR 的激活---------------------------------------------------------------------------07
1.2.3 AHR的配體----------------------------------------------------------------------------08
1.2.4 AHR對細胞貼附和遷移的相關調節----------------------------------------------08
1.2.5 AHR與MYCN關係 ----------------------------------------------------------------09
1.2.6 AHR 為神經母細胞瘤的良好預後因子------------------------------------------09
1.3 上皮間質轉化（EMT）---------------------------------------------------------------------11
1.3.1 介紹--------------------------------------------------------------------------------------11
1.3.2 EMT分為三種不同的亞型----------------------------------------------------------11
1.3.3 EMT與惡性腫瘤侵襲和轉移的關係----------------------------------------------12
1.3.4 常見的 EMT 標誌及作用-----------------------------------------------------------12
1.3.5 AHR調節EMT------------------------------------------------------------------------13
1.4 開發AHR 內源性配體的藥物-----------------------------------------------------------16
1.4.1 早期藥物--------------------------------------------------------------------------------16
1.4.2 中期藥物--------------------------------------------------------------------------------16
1.4.3 現今研究藥物--------------------------------------------------------------------------17
1.5 研究動機與目標-----------------------------------------------------------------------------18
第二章、材料與方法
2.1 RNA 提取-------------------------------------------------------------------------------------19
2.2 逆轉路------------------------------------------------------------------------------------------19
2.3 即時定量 PCR-------------------------------------------------------------------------------19
2.4 細胞遷移測定---------------------------------------------------------------------------------21
2.5 細胞貼附測定---------------------------------------------------------------------------------21
2.6 侵襲測定---------------------------------------------------------------------------------------22
第三章、實驗結果
3.1 化合物 Y 上調 CYP1A1的表達--------------------------------------------------------25
3.2 化合物 Y 抑制 SK-N-BE(2) 和 SK-N-SH 細胞的遷移和侵襲能力------------25
3.3 化合物 Y 抑制 SK-N-BE(2) 和 SK-N-SH 細胞的侵襲能力---------------------26
3.4 化合物 Y 抑制SK-N-SH 或 SK-N-BE(2)C 細胞的貼附能力-------------------26
3.5 化合物 Y 促進 E-cadherin 的表達上升和抑制 EMT 標誌物的表達----------26
3.6 化合物 Y 上調 KISS1 的表達----------------------------------------------------------27
第四章、討論------------------------------------------------------------------------------------------28
第五章、圖表
圖.1 化合物 Y 上調 CYP1A1 mRNA的表達-----------------------------------------------30
圖.2 化合物 Y 上調 AHR mRNA 的表達---------------------------------------------------31
圖.3 化合物 Y 抑制 SK-N-BE(2) 和 SK-N-SH 細胞的遷移---------------------------32
圖.4 化合物 Y 抑制SK-N-SH 或 SK-N-BE(2)C 細胞的侵襲-------------------------33
圖.5 化合物 Y 增加SK-N-SH 和 SK-N-BE(2)C細胞貼附能力-----------------------35
圖.6 化合物 Y 改變 EMT 不同標誌物 mRNA 的表達---------------------------------36
圖.7 化合物 Y 上調 KISS-1 mRNA 的表達------------------------------------------------37
第六章、參考文獻
6.1 Uncategorized References-------------------------------------------------------------------38

參考文獻

第六章、參考文獻
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指導教授

吳沛翊(Pei-yi Wu)

審核日期

2024-1-29

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