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研究生:吳宗儒
研究生(外文):Tzung-Ru Wu
論文名稱:探討因葉酸缺乏後所引起氧化壓力進而調控轉移相關基因表現以及強化轉移特性之機制研究
論文名稱(外文):Folate deficiency-induced enhancement of metastatic propensity is mediated by IOSS mechanism that
指導教授:劉燦榮劉燦榮引用關係
指導教授(外文):T. Z. Liu
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:59
中文關鍵詞:肝癌上皮-間質性態轉型胞內氧化壓力
外文關鍵詞:HCCEMTIOSS
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「肝癌」屬於世界十大癌症之一,以台灣本島而言,與肺癌同為主要癌症死因之首。一個有趣的現象指出肝癌末期病患普遍存有葉酸缺乏之共通點,60-85%肝癌病患血液檢體中即有此特色存在。而葉酸缺乏的環境培養下可促進增強黑色素瘤細胞之轉移特性。故此在本次研究計畫是要去釐清在葉酸缺乏環境培養下的肝癌細胞,是否能有意義的增強其轉移之特性。首先將pGSH-luc載體送入到SK-Hep-1細胞內永久性的表現並建立起此種細胞株(SKM),再分別培養在葉酸完全或缺乏之培養液中一至二週。發現除了葉酸缺乏後導致降低細胞生長並可誘導細胞變型為狀似纖維母細胞之型態。同時葉酸缺乏的細胞也能有明顯上升的氧化-硝化性壓力,推斷可能進而增強細胞內部侵犯轉移之特性。而在癌細胞的侵犯與移動的結果發現葉酸缺乏後較具有明顯的侵犯與爬行 行 為,同時也增強MMP-9活性,此外結果也透露出葉酸缺乏伴隨EMT系統之啟動:降低上皮細胞指標E-cadherin與增強間質細胞指標vimentin表現。進一步在活體內也證實葉酸缺乏具有高度轉移能力。總結以上之結果,推得當葉酸缺乏時能導致體內氧化-硝化壓力失去平衡,進而啟動EMT系統並刺激轉移相關基因表現,最後達成轉移之目的。
Hepatocellular carcinoma (HCC) is the first leading cause of cancer-related death in Taiwan. Although advances have been achieved in the diagnosis and treatment of HCC, the prognosis of patients with HCC remains dismal owing to the difficulty of controlling the metastasis. Interestingly, it has been observed that some nutritional surveys have reported low blood levels of folate in 60 to 85% of cases and folate deficiency also increased the metastatic potential of cultured melanoma cells. For this reason, we conduct this study for the purpose of shedding some lights on the question as to whether or not cancer cell cultivated under folate deficiency condition can promote their metastatic propensity. First we established stable cell line SKM that transfected with pGSH-luc empty. We found folate deficient-SKM cells exhibited lower proliferation and lesser adhension than supplemented condition. Flowcytometric data unequivocally indicated that SKM cells were being imposed with ROS and RNS than supplemented control. We further demonstrated that folate deficient-SKM cells were bestowed with enhanced invasive activity by increasing MMP-9 activity. Simultaneously, both migration and invasion were confirmed enhanced metastatic propensity of SKM cells. Additionally folate deficiency could render SKM cells to induce EMT promotion. Finally, we confirmed metastasis of in vitro results by xenograft animal model. Taken together, our data provide evidence that folate deficiency IOSS may play a pivotal factor in the promotion of metastatic propensity of HCC cells via modulating EMT candidate hallmarkers and metastasis related gene expressions.
指導教授推薦書……………………………………………………………………
口試委員會審定書…………………………………………………………………
授權書……………………………………………………………………………iii
誌謝………………………………………………………………………………iv
中文摘要……………………………………………………………………………v 英文摘要…………………………………………………………………………vi
縮寫表……………………………………………………………………………vii
目錄……………………………………………………………………………viii CHAPTER I. Introduction ……………………………………………………1
1.2 Metastasis…………………………………………………………………4
1.3 Folic acid status and ncer…………………………………………7
CHAPTER II. Specific Aim…………………………………………………9 CHAPTER III. Results………………………………………………………11 3.1 Folate deficiency causes growth retardation, but
promotes further mesenchymal transformation in SKM
cells………………………………………………………………………11
3.2 Folate deficiency promotes increased oxidative-
nitrosative stress via overproduction of reactive
oxygen species (ROS) and reactive nitrogen species (RNS)
………………………………………………………………………………12
3.3 Folate deficiency mediated oxidative-nitrosative stress
increases metastatic propensity via activating MMP-9
activity…………………………………………………………………13
3.4 Folate deficiency promoted EMT induction via
downregulation of E-cadherin and increased metastasis-
related protein expression………………………………………14 3.5 Folate deficiency enhanced metastatic propensity in day-
dependent manner in human tumor xenografts………………15 CHAPTER IV. Discussion……………………………………………………16 CHAPTER V. Material and Methods……………………………………21 3.1 Cell culture and generation of stable cell
lines………………………………………………………………………21
3.2 Cell viability assay………………………………………………21 3.3 Colony formation assay……………………………………………22 3.4 Flow cytometry analysis……………………………………………22 3.5 Zymogram analysis……………………………………………………23 3.6 Transwell migration assay…………………………………………23 3.7 Matrigel invasion assay……………………………………………24 3.8 Immunofluorescence and microscopy……………………………24 3.9 Western blotting………………………………………………………25 3.10 Animal model and diets……………………………………………26 3.11 Tumorgeniity assays in nude mice……………………………26 3.12 Xenogen IVIS bioluminescence imaging………………………27
CHAPTER VI. References……………………………………………………29 CHAPTER VII. Appendix and Figures……………………………………35
Appendix 1 Death rates per million population. ...........35
Appendix 2 The classical TNM staging system .............35
Appendix 3 Optical imaging of in vivo metastatic
process.......36
Appendix 4 Sites of EMT and MET in the emergence and
progression of carcinoma......................37 Fig. 1 Folate deficiency induced morphological change, loss
of cell-cell adhension and apical-basal polarity..
………………………………………………………………………… 38
Fig. 2 Folate deficiency impeded cell growth. However,
subsequent repletion of folate could render these
folate-starved cells to revive……………………………39
Fig. 3 Folate deficiency inhibited colony formation of SKM
cells and their restoration of colony formation
ability after folate resupplementation..……………40
Fig. 4 Folate deficiency induced S-phase
accumulation………………………………………………………41
Fig. 5 Folate deficiency induced overproduction of
endogeneous ROS and RNS and increased oxidative
stress in SKM cells……………………………………………42
Fig. 6 Folate deficiency promotes enhanced migration,
invasive propensity and increased MMP-9 activity..43
Fig. 7a Folate deficiency induced EMT candidate protein
expression, such as E-cadherin (E-CD), β-catenin (β-
CN) and vimentin (VN)…………………………………………44
Fig. 7b Folate deficiency induced angiogenic protein,
metastatic precursor and RNS, such as VEGF, RelA and
iNOS……………………………………………………………………45
Fig. 8 Folate deficiency induced EMT promotion and
angiogenesis…………………………………………..........46
Fig. 9 Folate deficiency enhanced metastatic propensity in
xenogrft mice model…………………………………………… 47
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