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研究生:Donia Alson
研究生(外文):Donia Alson
論文名稱:在小鼠模式中探討破傷風類毒素結合治療性癌症疫苗GVAX對抗子宮頸癌的機制
論文名稱(外文):Multiple Vaccination with Tumor-Cell Based Vaccine and its Effect in Combination with Tetanus Toxoid against Cervical Cancer in a Murine Model
指導教授:曲桐
指導教授(外文):S. C. Schuyler
學位類別:博士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2019
畢業學年度:108
語文別:英文
論文頁數:90
中文關鍵詞:no
外文關鍵詞:Cervical CancerHuman PappillomavirusImmunotherapyProphylactic VaccineGM-CSF
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Table of Contents
Recommendation Letter from the Thesis Advisor
Thesis/Dissertation Oral Defense Committee Certificate
Acknowledgement …………………………………………………… iii
Abstract ..…………………………………………………………….. iv Table of contents …………………………………………………….. vi
List of Figures ……………………………………………………….. ix
Chapter 1: Introduction ……………………………………………… 1
1.1 Cancer and Immune System …………………………………….. 1
1.2 Cancer Immunotherapy ………………………………………... 2
1.2.1 Immune Checkpoint Inhibitors …………………………….. 3
1.2.2 Adoptive T cell Therapy …………………………………… 4
1.3 Cervical Cancer and HPV ……………………………………... 5
1.3.1 HPV Genome ………………………………………………. 6
1.3.2 HPV Subtypes …………………………………………….. 9
1.3.3 HPV Viral Oncogenes ……………………………………… 9
1.3.4 HPV Infectious Cycle ………………………………………. 11
1.3.5 HPV Associated Cancer …………………………………… 12
1.3.6 HPV Vaccines……………………………………………….. 13
1.4 GVAX and GM-CSF …………………………………………… 14
1.4.1 Codon-Modified GM-CSF ………………………………….. 15
1.4.2 GM-CSF and Cancer………………………………………… 16
1.4.3 Anti-tumor mechanism of GM-CSF ………………………… 17
1.4.4 GM-CSF Monotherapy………………………………………. 18
1.5 Tetanus Toxoid and Immune Response ………………………… 19
1.6 Research Rational ………………………………………………. 20
1.7 Specific Aims …………………………………………………. 21
1.7.1 Experimental Design …………………………………….. 23
Chapter 2: Materials and Methods – I ……………………………… 24
2.1.1 Mice …………………………………………………………. 24
2.1.2 Cell Lines...……………………………………………………. 24
2.1.3 Tumor Model and Vaccination ……………………………… 25
2.1.4 Spleen Weight Index and Splenocyte Proliferation …………. 25
2.1.5 Cytokine Secretion Measurement with ELISA ……………… 26
2.1.6 Flow Cytometric Analysis of Immune Cells ………………… 26
2.1.7 In-Vivo Cytotoxicity Assay ……………………………….27
2.1.8 Statistical Analysis …………………………………………... 27
Materials and Methods – II
2.2.1 Mice ………………………………………………………….. 28
2.2.2 Cell Lines ……………………………………………………. 28
2.2.3 Cytokine Secretion Measurement with ELISA ........................ 29
2.2.4 Tumor Model and Vaccination ……………………………… 29
2.2.5 Flow Cytometric Analysis ………………………………….. 29
2.2.6 Statistical Analysis …………………………………………… 30
Chapter 3: Results – I
3.1.1 Higher level of GM-CSF secretion by TC-1/cGM-CSF stable cell line …………………………………………………………………… 31
3.1.2 Vaccination with tetanus toxoid and TC-1/cGM-CSF induced enhanced splenocyte proliferation …………………………………… 31
3.1.3 Increased levels of Th1 and Th2 cytokines in the group of mice vaccinated with combination vaccination …………………………… 32
3.1.4 Higher percentage of memory T cells generation after combination vaccination of tetanus and TC-1/cGM-CSF ……………………..…. 33
3.1.5 Generation of cytotoxic effector T cells in-vivo after combination vaccination with tetanus toxoid and TC-1/cGM-CSF ………….......... 34
3.1.6 Combination vaccination with tetanus toxoid and TC-1/cGM-CSF vaccine induced enhanced immunosurveillance and effectively inhibits tumorigenesis in vivo............................................................................. 35
Results – II
3.2.1 TC-1 cells transfected with LV-cGM-CSF(Lentiviral) (TC-1/cGM-CSF) expressed increased levels of GM-CSF compared with that of TC-1 cells transfected with LV-wtGM-CSF (TC-1/wtGM-CSF)……..…….. 36
3.2.2 Mice vaccinated with three doses of irradiated TC-1/cGM-CSF induce enhanced immunosurveillance compared with that of mice vaccinated for TC-1 tumor with one dose and five doses…………..….. 37
3.2.3 Generation of antigen-specific IFN-γ (Interferon-γ) producing CD8+ and CD4+ T cells …………………………………………………....… 38
3.2.4 A higher percentage of B220+ NK1.1+ interferon-producing killer dendritic cells (IKDCs) were produced in mice vaccinated with three doses of irradiated TC-1/cGM-CSF vaccine compared with that of mice vaccinated with one dose and five doses of irradiated TC-1/cGM-CSF vaccine………………………………………………………………… 39
Chapter 4: Discussion …………………………………………………. 41
Figures ………………………………………………………………… 49
References …………………………………………………………….. 62







List of Figures - I
Fig. 1. Increased levels of GM-CSF secretion by TC-1 cells containing codon-modified GM-CSF ………………………………………...….. 49
Fig. 2. Enhanced splenocyte proliferation after combination vaccination ……………………………………………………………………..….. 50
Fig. 3. Combination of tetanus toxoid and TC-1/cGM-CSF vaccination enhances the level of Th1 and Th2 cytokine secretion …………..….. 51
Fig. 4. Combination vaccination increase the percentage of CD8+CD44+ memory T cell lymphocyte in the spleen ……………………….…. . 52
Fig. 5. Combination vaccination increase the percentage of CD4+CD44+ and CD8+CD44+ memory T cell lymphocyte infiltration in the tumor …………………………………………………………………….…. 53
Fig. 6. Effects of combination vaccination on the generation E7-specific CTL in C56BL/6 mice …………………………………………….… 54
Fig. 7. Combination vaccination with tetanus toxoid and TC-1/cGM-CSF can effectively inhibit tumor growth ………………………………. 55










List of Figures – II
Fig 8. Increased levels of GM-CSF production by TC-1 cells containing codon-modified GM-CSF……………………………………………... 56
Fig 9. Tumor vaccination three-times with cGM-CSF can efficiently inhibit tumor growth compared to five-time vaccination…………..….. 57
Fig 10. Vaccination three-times with cGM-CSF generates a higher IFN-γ secreting CD8+ T and CD4+ T cell response compared to five-times….. 59
Fig 11. Vaccination three-times with irradiated TC-1/cGM-CSF vaccine generates a higher percentage of IKDCs compared to vaccination five-times……………………………………………………………………. 61
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