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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82500完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蘇剛毅(Kang-Yi Su) | |
| dc.contributor.author | Cheng-Fang Wu | en |
| dc.contributor.author | 吳承芳 | zh_TW |
| dc.date.accessioned | 2022-11-25T07:45:50Z | - |
| dc.date.available | 2023-08-31 | |
| dc.date.copyright | 2021-09-16 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-18 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/82500 | - |
| dc.description.abstract | "腫瘤生成(tumor initiation)為基因變異所致，癌細胞中的基因突變可大致分成驅動突變(driver mutations)及乘客突變(passenger mutations)，其中驅動突變具有生長優勢，可不受正常細胞增殖、分化等限制，持續擴增成癌細胞，帶有此特性的基因稱作癌症驅動基因(cancer driver genes)。在非小細胞肺癌中，約有2/3病人帶有驅動基因變異，在表皮生長因子受體(epidermal growth factor receptor, EGFR)變異中，又以exon 19 deletion或是exon 21 L858R mutation所占比例最高。粒線體與癌症有密切關聯，已有研究指出細胞在腫瘤生成時會發生代謝途徑重組(metabolic reprogramming)而與正常細胞有所差異，可做為治療標的。因此，我們想了解在突變型EGFR驅動的腫瘤生成，有哪些粒線體蛋白參與在細胞代謝的改變。 首先，在NIH3T3細胞株過表現EGFR L858R篩選出穩定表現的細胞，並確認這些細胞有較好的細胞增生及群落形成能力，已有研究指出EGFR突變之癌細胞會使其下游蛋白表現上調，但在我們的細胞模型及動物模型這些下游蛋白並沒有顯著改變，因此，我們推測腫瘤生成時細胞會先改變細胞代謝讓自己增生不經由典型的癌細胞EGFR訊息傳遞。為了進一步了解哪些蛋白與細胞代謝改變有關，我們萃取NIH3T3過表現EGFR L858R及對照組的粒線體蛋白進行蛋白質體(proteome)分析，根據結果挑選差異倍數(fold change)大於1.5及小於0.67之蛋白，分析其路徑多與細胞週期及代謝相關，我們也在氧化磷酸化(oxidative phosphorylation)及糖解(glycolysis)功能性實驗中，發現EGFR L858R組別在這兩種代謝途徑的使用與對照組相比皆有顯著差異。 同時，在過表現EGFR L858R之細胞及帶有轉殖基因(transgene)的動物模型中，皆有看到一部分EGFR L858R轉位(translocation)到粒線體的情形且正常細胞與癌細胞的蛋白分子量有所差異，因此，我們推測轉位到粒線體的EGFR L858R可能與其他蛋白作用，參與細胞代謝改變，未來將更進一步探討此交互關係與機制。" | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-25T07:45:50Z (GMT). No. of bitstreams: 1 U0001-1808202113262500.pdf: 5146210 bytes, checksum: beb0f7394a4bb0c3189ebe969c2a88bc (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | 口試委員審定書 i 致謝 ii 中文摘要 iii Abstract iv Chapter 1 Introduction 1 1.1 Introduction of tumorigenesis 2 1.2 Cancer driver genes (CDGs) 2 1.3 Mutant epidermal growth factor receptor (EGFR) is one of the most frequently driver in non-small-cell lung cancer (NSCLC) 3 1.3.1 Introduction of lung cancer 3 1.3.2 EGFR mutations and EGFR tyrosine kinase inhibitor (EGFR-TKI) 3 1.4 Mitochondria plays the crucial role during tumorigenesis 4 1.4.1 Fission and fusion dynamics 4 1.4.2 Bioenergetics 5 1.4.3 Oxidative stress 5 1.4.4 Metabolism 6 Chapter 2 Specific aim 8 Chapter 3 Material and Method 10 3.1 Cell culture 11 3.2 Plasmid construction 11 3.3 Transfection and stable cell line establishment 12 3.4 Transgenic mouse models 13 3.5 Genotyping 13 3.6 Tumor induction 14 3.7 Pathology interpretation 14 3.8 Protein extraction and Western blotting analysis 14 3.9 RNA extraction and real-time quantitative PCR (qPCR) 15 3.10 MTT assay 16 3.11 Colony formation assay 17 3.12 Bioenergetic Analysis of Oxygen Consumption Rate (OCR) 17 3.13 Lactate assay 18 3.14 Mitochondrial fractionation 18 3.15 Immunofluorescence microscopy 18 3.16 Proteomic 19 3.17 Statistical analysis 20 Chapter 4 Result 21 4.1 Establishment of NIH3T3 stable cell lines with EGFR L858R expression 22 4.2 Overexpression of EGFR L858R promotes cell transformation of NIH3T3 cells 23 4.3 Overexpression of EGFR L858R in normal cell may through non-canonical EGFR signaling pathway of cancer cells 23 4.4 Metabolism-related pathways enriched in EGFR L858R-driven tumor initiation by mitochondrial proteomics 25 4.5 Enhanced oxidative phosphorylation and glycolysis of NIH3T3-EGFR L858R 26 4.6 EGFR L858R translocated to mitochondria in EGFR L858R-driven tumor initiation 27 Chapter 5 Conclusion 28 Chapter 6 Discussion 30 6.1 Kinase-independent (KID) functions of EGFR 31 6.2 EGFR-induced metabolic reprogramming during tumorigenesis 31 6.3 EGFR L858R translocated to mitochondria 32 Chapter 7 Figure 34 Figure 1. Validation of pCMV-Tag2A-EGFR L858R plasmid 35 Figure 2. EGFR L858R overexpression induces cell transformation of NIH3T3 cells 38 Figure 3. Western blotting of NIH3T3-EGFR L858R and CL1-0-EGFR L858R stable clones 39 Figure 4. Western blotting of transient expression EGFR L858R in CL1-0 and NIH3T3 cell lines 40 Figure 5. Phenotype of transgenic mice 42 Figure 6. Western blotting of mouse lung tissue lysate 43 Figure 7. Gene ontology analysis of NIH3T3 mitochondria proteomics 46 Figure 8. Gene ontology analysis of FC ≥ 1.5 non-mitochondrial protein and FC ≤ 0.67 mitochondrial protein from NIH3T3 mitochondria proteomics 49 Figure 9. Oxidative phosphorylation and glycolytic metabolism in NIH3T3-vector and NIH3T3-L858R cells 53 Figure 10. EGFR L858R translocated to mitochondria in both in vitro and in vivo models 55 Chapter 8 Table 56 Table 1. List of antibodies 57 Table 2. List of primers 59 Chapter 9 Reference 60 Chapter 10 Appendix 67 Appendix 1. Transgenic mice model with inducible lung-specific EGFR L858R 68 | |
| dc.language.iso | en | |
| dc.title | 探討突變型EGFR透過粒線體代謝轉變促進腫瘤生成 | zh_TW |
| dc.title | Study of Activating Mutant EGFR Driven Tumor Initiation through Mitochondria-Mediated Metabolism Shift | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林亮音(Hsin-Tsai Liu),楊雅倩(Chih-Yang Tseng),郭靜穎 | |
| dc.subject.keyword | 腫瘤生成,EGFR L858R,粒線體,代謝,蛋白質體,轉位, | zh_TW |
| dc.subject.keyword | tumor initiation,EGFR L858R,mitochondria,metabolism,proteome,translocation, | en |
| dc.relation.page | 68 | |
| dc.identifier.doi | 10.6342/NTU202102465 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-08-19 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| dc.date.embargo-lift | 2023-08-31 | - |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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