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系統識別號 U0026-1708201414342000
論文名稱(中文) 在小鼠原位膀胱腫瘤模式中自噬作用抑制膀胱腫瘤的生成
論文名稱(英文) Autophagy suppresses bladder tumor formation in a mouse orthotopic bladder tumor formation model
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 102
學期 2
出版年 103
研究生(中文) 郭琬婷
研究生(英文) Wan-Ting Kuo
學號 s46011061
學位類別 碩士
語文別 英文
論文頁數 50頁
口試委員 指導教授-劉校生
口試委員-周楠華
口試委員-張志鵬
口試委員-劉怡文
中文關鍵字 自噬作用  原位癌模式  膀胱癌 
英文關鍵字 Autophagy  orthotopic tumor model  bladder cancer 
學科別分類
中文摘要 自噬作用是一個細胞分解的過程，當細胞受到壓力刺激下，此過程會包裹一些不需要或受損的細胞質內的物質，接著利用溶小體的機制進行分解。自噬作用在腫瘤生成過程中會透過清除致癌蛋白等方式來抑制腫瘤生長。這顯示自噬作用可作為治療癌症的方法。因此尋找具有潛力誘導自噬反應的藥物是一個創新的抗癌策略。X藥物是臨床上廣為使用的抗心律不整藥物，最近被發現可以誘導自噬反應。我們的in vitro研究顯示X藥物在老鼠和人類的膀胱癌細胞中誘發自噬反應，而且癌細胞株和腫瘤的生長都有被抑制。我們進一步建立帶有冷光基因表現的老鼠膀胱癌細胞株 (MB49Luc)。將此細胞株植入C57BL/6母鼠的膀胱表皮上，利用非侵入式活體分子影像系統追蹤冷光表現，觀察腫瘤生長情形。研究結果顯示在膀胱灌流X藥物的組別當中，冷光的表現隨著時間的增加而減少，顯示腫瘤有變小的情形。透過Hematoxylin和eosin組織染色更進一步證實腫瘤生成受到抑制。利用免疫化學染色法和免疫螢光染色的方式觀察到在膀胱灌流X藥物的組別當中，小鼠的膀胱組織有明顯自噬現象產生。在X藥物的組別裡，對小鼠生理造成的副作用很低。此外也比較X藥物與臨床上治療膀胱癌藥物mitomycin C兩者抑制腫瘤的效果和其副作用。結果顯示X藥物和臨床用藥mitomycin C都具有抑制腫瘤生成，我們也分析小鼠生理現象，mitomycin C有著明顯的副作用產生。總而言之，我們在in vitro和in vivo實驗中證實X藥物可以抑制膀胱腫瘤生成透過自噬反應。在臨床上治療膀胱癌病人的藥物當中，我們發現X藥物是一個新的非標靶和低副作用的抗癌藥物。
英文摘要 Autophagy is a catabolic process which involves in recruitment of unnecessary and damaged proteins and cellular components through degradation in the lysosomal machinery under stress conditions. Autophagy plays a suppressive role in tumorigenesis through the degradation of oncoprotein. Autophagy may be a potential therapy for cancer. Therefore, to find out a potential autophagy inducer drug as therapy route is needed. X drug which is an antiarrhythmic drug was identified as an autophagy inducer. Our in vitro study showed that X drug can induce autophagy and inhibit proliferation and colony formation of the MB49 mouse bladder cancer cells and the T24 human bladder cancer cells. The mouse bladder cancer cell lines named MB49Luc stably expressing the luciferase gene were further established. Orthotopic bladder tumor formation in C57BL/6 female mice after MB49Luc cell inoculation was further established and monitored by tracing bioluminescence expression under the IVIS imaging system. Our results show that the bioluminescence expression accompanied with the tumor size of the treated mice was decreased after intravesical instillation of X drug. We further confirm that bladder tumor was inhibited by hematoxylin and eosin stain. X drug -induced autophagy in the bladder tissue of the treated mice was confirmed by immunohistochemistry and immunofluorescence assays. X drug showed low side effect on the physiologic conditions of the treated mice. Furthermore, comparing the toxicity and efficacy of tumor suppression between X drug and clinical used anti-bladder cancer drug mitomycin C. X drug and mitomycin C both show tumor suppression capability. We also analyze the physiologic conditions of the treated mice. Our results show that mitomycin C has significant side effect in the treated mice. In summary, intravesical delivery of X drug suppresses tumorigenesis of bladder cancer cells both in vitro and in vivo through activation of autophagy. In the clinical therapeutic agents of bladder cancer patients, we reveal that X drug is a novel off-labeling and low side effect agent.
論文目次 中文摘要 I
Abstract II
Acknowledgement IV
Abbreviation VIII
Introduction
I.Bladder cancer 1
II.Orthotopic murine bladder tumor model 1
III.Autophagy 2
IV.Autophagy and tumorigenesis 3
V.X drug 4
Materials and Methods
I.Cell lines and cell culture 6
II.Murine orthotopic bladder tumor model 6
III.Preparation of protein of mice tissue 8
IV.Immunofluorescent staining 8
V.Immunohistochemical staining 8
VI.Transabdominal Micro-Ultrasound Imaging 9
VII.IVIS Spectrum 9
VIII.Western blotting 10
IX.Cell proliferation 10
X.Colony formation assay 11
XI.Luciferase reporter assay 11
XII.Statistical Analysis 11
Results
I.X drug induces autophagy and inhibits tumor growth in mouse and human bladder cancer cell lines 13
II.A model of mouse orthotopic bladder tumor formation was established 13
III.The mouse bladder cancer cell lines MB49Luc which stably express luciferase reporter gene were established 14
IV.Tumor formation of the stable MB49-Luc cells in bladders of C57BL/6 female mice using IVIS system 15
V.X drug suppresses bladder tumor formation in vivo 15
VI.X drug induces autophagy in mucosa of bladder tissues in vivo 16
VII.X drug showed low side effect on the physiological parameters of the untreated mice 16
VIII.Transabdominal micro-ultrasound imaging was conducted to monitor the tumor formation and tumor mass in the murine bladder tumor model 17
IX.Both X drug and mitomycin C suppress bladder tumor formation but mitomycin C showed side effect on physiology of the mice 18
Discussion 20
References 24
Curriculum Vitae 50
Table and Figure List
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