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研究生:林士文
研究生(外文):Shr-Uen Lin
論文名稱:以活體功能喪失篩選平台尋找腫瘤轉移抑制基因
論文名稱(外文):Identification of Tumor Metastatic Suppressors by in vivo Loss-of-Function Screening System
指導教授:俞松良俞松良引用關係
口試委員:李財坤詹迺立蘇剛毅
口試日期:2013-06-21
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學檢驗暨生物技術學研究所
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:2013
畢業學年度:101
語文別:英文
論文頁數:66
中文關鍵詞:癌轉移次世代定序組織切片培養
外文關鍵詞:tumor metastasisnext generation sequencingorganotypic culture
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隨著醫療的進步,除腦癌及淋巴癌外,多數原發性腫瘤能有效的被控制。但是一旦發生轉移,通常會在許多組織器官中出現,無法完全清除進而造成器官衰竭甚至個體死亡,統計結果顯示,90%因癌症而過世的人有發生癌轉移。近年來實驗儀器技術進步,使我們可以高通量去研究分析各個基因的功能,在本篇研究中,我透過基因體核糖核酸干擾方式,將82,000株帶有不同shRNA質體的lentivirus感染穩定表現螢火蟲冷光酵素的A549肺腺癌細胞並打入NOD/SCID老鼠左肺葉中,以IVIS追蹤記錄腫瘤的成長及轉移。以次世代定序儀分析各個轉移腫瘤中所帶有的shRNA序列及所占的比例。發現從三隻老鼠的八顆轉移腫瘤中,相較於左肺葉中注入的腫瘤,有64個shRNA 質體所占的比例超過百倍的增加。此外,不論是原發或轉移的腦癌都極為致命,其浸潤式顱內轉移在治療上相當困難,因為目前針對癌症轉移尚缺乏精準的實驗模式,我嘗試設計或優化現有的癌症研究模式,希望能針對癌轉移各個步驟去做定量觀察:包括原位注射後,並透過IVIS數據試圖重建腫瘤體積及位置;同時也將小鼠頭蓋骨去除,直接用顯微鏡觀察,以達到活體、單一細胞等級的觀察;同時從小鼠頸動脈注入細胞,去觀察腫瘤細胞通過血腦障壁及在腦部形成腫瘤的能力;最後,培養初生小鼠腦部活組織切片,並在上面種上腫瘤細胞,提供一個保有組織結構、細胞組成、微環境,且可以長時間、多次、高解析度的實驗方式,來研究腦癌的轉移及增生。

Most of primary tumors can be controlled by surgical excision. However, systemic metastasis and organs failures often cause individual death. More than 90% of people died from cancer had developed metastasis. With advanced high through put tools we are now able to screen human genome for specific cell functions. In this study we applied genome-wide RNA interference to find novel tumor metastatic suppressors. Firefly luciferase-expressing A549 lung adenocarcinoma cells were stably transfected with lentivirus based shRNA library and then orthotopically injected into left lobes of NOD/SCID mice. The proliferation and metastasis of inoculated tumors were traced by IVIS. Analysis of shRNA sequences in harvested metastatic tumors was accomplished by next generation sequencing. 64 shRNAs were enriched greater than 100-fold in eight metastatic tumors from three mice. Regarding to intracranial tumors, they are fatal no matter their origins. The infiltrative invasion nature of intracranial tumors limits surgical therapy and is still hard to study. Thus we try to develop 4 experimental models to focus on different steps during metastasis. First we established the reconstruction of tumor volume from IVIS data of brain tumor orthotopic model and direct observation brain tumors of skull stripped mice for in vivo single cell resolution assay. We also developed an intracarotid artery injection model in which tumor cells were injected into intracarotid artery and the colonies formed in brains were recorded. Last, we organotypically cultured brain slices of neonatal mice providing well-preserved tissue architecture and microenvironment to tumor cells and measured brain tumor proliferation and invasion by time-lapse and high resolution microscopy.

口試委員審定書……………………………………………………………………I
Acknowledgements…………………………………………………………………II
中文摘要……………………………………………………………………………III
Abstract……………………………………………………………………………IV
Chapter 1 Introduction……………………………………………………………1
1.1 Cancer…………………………………………...…...……………………….…2
1.2 Metastasis…………………………………………………………….............2
1.3 Brain cancer……………………………………………………………………3
1.4 Lung cancer…………………………………………………………………….4
1.5 shRNA library…………………………………………………………………5
1.6 Cancer and microenvironment……………………….………………………5
1.7 Orthotopic tumor implantation……………………………………………6
1.8 Organotypic tissue culture……………………………………………………..7
Chapter 2 Objectives………………………………………………………………9
2.1 Identification of Lung Cancer Metastatic Suppressors by in vivo shRNA Screening.……………………………………………………………………10
2.2 Quantification of proliferation, invasion, and tumorigenesis of glioma..…10
Chapter 3 Materials and Methods………………………………………………11
3.1 Cell lines and culture conditions……………………………………………..12
3.2 Lentivirus based shRNA library and transduction…………………………12
3.3 Mice…………………………………………………………………………….13
3.4 Brain orthotopic injection……………………………………………………..13
3.5 Skull windowing assay………………………………………………………..14
3.6 Left lung lobe orthotopic injection…………………………………………...14
3.7 In vivo Imaging System………………………………………………………15
3.8 Animal sacrifice and tumor cells culture…………………………………….15
3.9 Genome DNA extraction……………………………………………………..16
3.10 PCR amplification and DNA gel extraction………………………………17
3.11 Next generation sequencing and data analysis…………………………18
3.12 Slice preparation……………………………………………………………19
3.13 Live/Dead stain……………………………………………………………19
3.14 Seeding tumor cells on brain slices………………………………………20
3.15 Tumor seeding……………………………………………………………...21
3.16 Carotid artery injection……………………………………………………..21
Chapter 4 Results…………………………………………………………………23
4.1 Successful lung orthotopic tumor injection and traced by IVIS…………..24
4.2 Tumor-bearing mice mimic clinical patients………………………………..24
4.3 Cell culture from primary and metastasis tumors…………………………25
4.4 NGS and data processing……………………………………………………26
4.5 Skull windowing mice is a powerful model for real time observation….27
4.6 Brain slice preparation………………………………………………………..28
4.7 Buffer and medium test………………………………………………………28
4.8 Supplement optimization……………………………………………………..29
4.9 Seeding tumor cells on the slices…………………………………………..30
4.10 Quantification of cell invasiveness………………………………………...30
4.11 Intracarotid tumor cell injection formed tumors in brain………………….31
Chapter 5 Figures and Tables……………………………………………………33
Chapter 6 Discussion………………………………………………………………54
6.1 Combine genome-wide human RNAi and lung orthotopic tumor model...55
6.2 Applications of carotid artery injection………………………………………56
6.3 Validation by multiple assays of brain metastasis…………………………57
6.4 Brain slice for neural toxicity test…………………………………………….58
Chapter 7 References…………………..…………………………………………59


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