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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蘇剛毅 | |
| dc.contributor.author | Yun-Ting Hsieh | en |
| dc.contributor.author | 謝昀庭 | zh_TW |
| dc.date.accessioned | 2021-06-15T12:39:19Z | - |
| dc.date.available | 2018-08-26 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-07-28 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50403 | - |
| dc.description.abstract | 腫瘤浸潤淋巴球已知存在於包含肺癌的多種癌症中。參與後天免疫的淋巴球成熟過程所產生的可變、變異、連接片段的重組，構成免疫全面多樣性。透過免疫全面多樣分析，可以用於監測個體的T細胞受體與B細胞受體在疾病中的群殖增生。T細胞受體與抗原的互補決定區(CDR)最具變異性的區域為CDR3，本篇研究假設其會在突變型表皮生長因子受體所誘發的肺癌中受到特殊腫瘤抗原刺激而進行群殖增生，其增生頻譜可望用於新穎抗原依賴的免疫療法。首先，利用雙基因轉殖鼠搭配可誘導轉位子系統，將帶有肺組織特異性的CC10基因，及具酪胺酸激酶抑制劑敏感性的突變型表皮生長因子受體EGFR L858R或具酪胺酸激酶抑制劑抗藥性的EGFR L858R+T790M轉殖入C57BL/6品系小鼠，以含有625ppm四環黴素飼料餵食小鼠八周後，帶有EGFR轉殖基因的實驗組小鼠肺高度表達EGFR而誘導肺腺癌；而在餵食四周後以Iressa(一種酪胺酸激酶抑制劑)治療小鼠至十六周，癌細胞會大幅減少。以流式細胞儀分析肺沖洗液，顯示CD4+輔助性T細胞比例大於CD8+胞殺性T細胞;EGFR L858R+T790M轉殖小鼠的CD4+ T細胞中CD4+調節性T細胞多於EGFR L858R轉殖小鼠。接著，利用次世代定序分析肺臟、脾臟、全血中T細胞CDR3的cDNA，用以區分癌細胞周圍的TCR群殖增生以及全身性的分布。以GeneSpring軟體分析T細胞 CDR3的定序讀值數，發現在帶有肺腺癌小鼠的肺臟有TCR CDR3的群殖增生，而在脾臟則呈現離散型態。肺臟增殖最多的前十名T細胞 CDR3片段序列帶有部分胺基酸的相似性，顯示其可能源自於某種特定的肺癌抗原。結果顯示具有肺癌特異性的T細胞CDR3可能因特定抗原而在肺臟群殖增生，在全面性免疫系統中則無。在誘導表皮生長因子受體以及以Iressa治療後，小鼠全血可用於監測血液TCRβ增值變化，便於作為生物指標。另外，也期待免疫全面策略可成為新的EGFR誘導型肺癌免疫療法，甚至用於尋找酪胺酸激酶抑制劑抗藥性相關的抗原。 | zh_TW |
| dc.description.abstract | Tumor infiltrating lymphocytes (TILs) are implicating to deal with tumor cells, including lung tumors. Immune repertoire, the overall V(D)J rearrangements in lymphocytes, is a potential index to monitor individual’s T cell receptors (TCRs) or B cell receptors (BCRs) clonal expansion in cancer progression and remission. We hypothesized the TCRβ CDR3, the most various region of antigen binding site, would be functionally and specifically expand in EGFR-driven lung cancer due to tumor antigens stimulation or immune-editing. In this study, we established a mice model to feature a TIL-TCRβ expanding profile in lung cancer.
First, Tet-On system was applied to the bi-transgenic mice bearing Clara cell promotor CC10 plus mutant EGFR L858R (EGFR TKI sensitive) or mutant EGFR L858R+T790M (EGFR TKI resistance). With 625ppm doxycycline (tetracycline analogue) induction for lung EGFR expression, mice lungs highly expressed adenocarcinoma. In contrast, lung tumors remission occurred with Iressa (EGFR-Tyrosine kinase inhibitor, TKI) administration. Flow cytometry analysis of BALF identified higher CD4+/CD3+ T cells ratio in lungs, and more CD4+ regulatory T cells in lungs of EGFR L858R+T790M mice than EGFR L858R mice. Then, we used next generation sequencing (NGS) to analyze cDNA of the TCRβ CDR3 in lungs and spleens. Total reads of TCRβ CDR3 represented the abundance of expanded clones. The results suggested some expanded TCRβ CDR3 clones in lung but distributed clones in spleen. The top abundant lung TCRβ CDR3 sequences have similar amino acids order, pointing that these may result from specific antigens. Hence, we claimed tumor-specific TCRβ CDR3 would clonally expand in tumor site but not in overall immune system. To clarify whether TCRβ CDR3 would expand as a profile in peripheral blood, whole blood samples will be analyzed. With the whole immune-profile, we expected TCRβ CDR3 as a potential biomarker of cancer progression and remission, and as an indication of novel lymphocyte-dependent immune therapy. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T12:39:19Z (GMT). No. of bitstreams: 1 ntu-105-R03424001-1.pdf: 2959992 bytes, checksum: fe00c63e5eb24103380c5d581c1b9306 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | Part I
Analysis of TCRβ CDR3 Profiles of Mutant EGFR-Driven Lung Adenocarcinoma 中文摘要 2 Abstract 4 1. Introduction 6 1.1 Lung cancer 7 1.1.1 Lung cancer epidemiology 7 1.1.2 Lung cancer category 7 1.1.3 Treatment for each stage of lung cancer 8 1.1.4 Mutational category of lung cancer and target therapy 9 1.1.5 Limitations of target therapy 10 1.1.6 Development of immune therapy in lung cancer 11 1.1.7 Limitations of immune therapy 12 1.1.8 Unmet goals of lung cancer diagnosis and therapies 13 1.2 Lung cancer immunology 14 1.2.1 Immunophenotypes of lung cancer 14 1.2.2 Immune repertoire 16 1.2.3 Immune repertoire analysis 17 1.3 Rationale 18 2. Specific aim 19 3. Materials and methods 21 3.1 Transgenic mice model 22 3.2 Genotyping and PCR 22 3.3 Experimental treatment of doxycycline and EGFR-TKI 23 3.4 Pathology interpretation 24 3.5 Protein extraction and Western blotting 24 3.6 TCR library preparation 25 3.7 Processing of genome analyzer sequence data 26 3.8 Identifying CDR3 sequences 27 3.9 Comparisons of CDR3 sequences between control and experimental groups 27 3.10 Flow cytometry of bronchoalveolar lavage fluid and splenocytes 27 4. Results 29 4.1 Experimental design 30 4.2 Transgenic EGFR L858R mice is more sensitive to doxycycline induction of EGFR than EGFR L858R+T790M mice 30 4.3 Immunophenotype of lung cancer 31 4.4 Proportion of T cell subsets in bronchoalveolar lavage fluid (BALF) 32 4.5 Proportion of T cell subsets in spleen 34 4.6 Lung and spleen TCRβ CDR3 sequence profiling of CC10 and EGFR L858R mice 34 4.7 Clonal expansion of TCRβ CDR3 in lungs of mice with adenocarcinoma 35 4.8 NGS of blood samples 36 5. Discussion 37 5.1 Application of TCRβ CDR3 analysis between groups 38 5.2 Depletion the concern of toxicity and immune alteration from doxycycline and Iressa treatment 38 5.3 Transgene EGFR L858R+ T790M represents germline TKI-resistant mechanism which is different from clinical patients 39 5.4 Determination of heterogeneity and protein profiles in transgenic EGFR mutation mice model 40 5.5 T cells activation and suppression in lung cancer 40 5.6 Increased CD3+CD8-CD4- T cells in adenocarcinoma stage 41 5.7 Upregulated CD4+ T cells in severer lung cancer and numerous CD4+ Treg in EGFR L858R+T790M mice 42 5.8 Proportion of CD8+ and CD4+ splenocytes increased in EGFR L858R mice from 4 weeks to 8 week induction 43 5.9 Samples failed in NGS are not qualified to analyze 44 5.10 Non- identical TCR CDR3 sequence clonal expand in lung and spleen 44 5.11 Limitations of TCRβ CDR3 sequencing 45 5.12 Advanced tools for TCR analysis 46 5.13 T-cell inhibitory molecules expression in lung cancer 47 6. Figures 49 Figure 1. Experimental design 50 Figure 2. Mice model confirmation 52 Figure 3. Immuno-regulation in lung tumor microenvironment 53 Fig4. Rich proportion of macrophages but few T cells in bronchoalveolar lavage fluid (BALF) at 0 weeks EGFR induction 54 Fig5. CD3+ cells character in bronchoalveolar lavage fluid (BALF) 55 Fig6. Proportion of T cell subsets in splenocytes 56 Figure 7. Depletion of lung TCRβ CDR3 sample during processing 57 Figure 8. Spleen and lung TCRβ CDR3 similarity analysis by GeneSpring 58 Figure 9. Sequences of TCRβ CDR3 clonal expand in lung tumor 59 7. Tables 60 Table 1. Mice spleen and lung TCRβ CDR3 total reads number of next-generation sequencing 61 Table 2. Rank of clonal expanded Lung TCRβ CDR3 during lung cancer progression 62 Table 3. Different time points of whole blood total reads number from next generation sequence of L858R mice 63 Part II The Effects of High Fat Diet on Mutant EGFR-Driven Lung Adenocarcinoma 64 中文摘要 65 Abstract 66 1. Introduction 67 1.1 Epidemiology of overweight and obesity 68 1.2 Obesity: definition and causes 68 1.3 Obesity and chronic disease 69 1.4 Obesity and cancers 70 1.5 Rationale 71 2. Specific aim 72 3. Material and methods 74 3.1 Mice model 75 3.2 High fat diet treatment and EGFR induction 75 3.3 Pathology interpretation 76 3.4 Western blotting 76 3.5 Statistical analysis 77 4. Results 78 4.1 Phenotype of high fat diet-fed mice with EGFR induction by turns for 10 weeks 79 4.2 Body weights varied depending on diet 79 4.3 Expression of EGFR signaling molecules of lung cancer with rotated high fat diet 80 4.4 Phenotype of high fat diet-fed mice combined with EGFR induction for 7 weeks 80 4.5 Body weight loss rapidly at late stage of adenocarcinoma with high fat diet 81 4.6 High fat diet enhanced EGFR phosphorylation but decreased AKT phosphorylation 81 4.7 Less AKT de-phosphorylation of HFD treated mice with early stage of EGFR induction 82 5. Discussion 83 5.1 Insufficient difference of lung adenocarcinoma between high fat diet-fed mice and regular diet fed mice by rotating with doxycycline diet 84 5.2 Great variation of lung weight within high fat diet-fed mice 84 5.3 Adiponectin may contribute to AKT de-phosphorylation in long term HFD mice 85 5.4 Conclusion and future prospects 87 6. Figures 89 Fig 1. Phenotype of lung cancer induction rotated with high fat diet 90 Fig 2. Normalized body weight of mice which had been induced lung cancer by rotating high fat diet and regular diet 91 Fig 3. Western blotting of EGFR signaling pathway in EGFR-induced lung cancer by rotating with high fat diet or regular diet 92 Fig 4. Phenotype of EGFR-driven lung cancer with high fat diet treatment 93 Fig 5. Normalized body weight of mice with EGFR-driven lung cancer under high fat diet treatment and regular diet treatment 94 Fig 6. Western blotting of mice with EGFR-driven lung cancer under high fat diet treatment and regular diet treatment 95 Fig 7. Comparison of EGFR and its downstream between 4.5 weeks and 7 weeks EGFR induction in HFD mice 97 7. References 98 | |
| dc.language.iso | en | |
| dc.subject | 次世代定序 | zh_TW |
| dc.subject | 免疫全面多樣 | zh_TW |
| dc.subject | T細胞受體互補決定區3 | zh_TW |
| dc.subject | 酪胺酸抑制劑抗藥性 | zh_TW |
| dc.subject | 次世代定序 | zh_TW |
| dc.subject | 突變型表皮生長因子受體L858R誘導肺腺癌 | zh_TW |
| dc.subject | 免疫全面多樣 | zh_TW |
| dc.subject | T細胞受體互補決定區3 | zh_TW |
| dc.subject | 酪胺酸抑制劑抗藥性 | zh_TW |
| dc.subject | 突變型表皮生長因子受體L858R誘導肺腺癌 | zh_TW |
| dc.subject | TKI-resistance | en |
| dc.subject | T cell receptor CDR3 | en |
| dc.subject | immune repertoire | en |
| dc.subject | NGS | en |
| dc.subject | EGFR L858R-driven lung adenocarcinoma | en |
| dc.subject | TKI-resistance | en |
| dc.subject | T cell receptor CDR3 | en |
| dc.subject | immune repertoire | en |
| dc.subject | NGS | en |
| dc.subject | EGFR L858R-driven lung adenocarcinoma | en |
| dc.title | T細胞受體β鏈互補決定區3之頻譜暨高脂飲食研究於突變型表皮生長因子受體誘發之肺腺癌小鼠 | zh_TW |
| dc.title | TCRβ CDR3 Profile and High Fat-diet Study on Mutant EGFR-Driven Lung Adenocarcinoma Mice Model | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林亮音,俞松良,楊雅倩 | |
| dc.subject.keyword | T細胞受體互補決定區3,免疫全面多樣,次世代定序,突變型表皮生長因子受體L858R誘導肺腺癌,酪胺酸抑制劑抗藥性, | zh_TW |
| dc.subject.keyword | T cell receptor CDR3,immune repertoire,NGS,EGFR L858R-driven lung adenocarcinoma,TKI-resistance, | en |
| dc.relation.page | 132 | |
| dc.identifier.doi | 10.6342/NTU201601574 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-07-28 | |
| 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 |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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