臺灣博碩士論文加值系統

子宮頸癌所造成的死亡人數是全球女性因癌症死亡人數排名的第二位，致癌途徑是經由幾種高危險型的人類乳突病毒 (human papillomaviruses, HPV)在人體內進行持續性感染所導致。近年來，儘管針對HPV第16,18型的預防性疫苗已經核可問世並獲得不錯的預防成效，醫學上仍需要治療型的疫苗來對抗那些曾遭高危險型HPV持續性感染的婦女。為了提升治療型的HPV疫苗產生的抗原呈獻能力，在先前的研究中，我們將老鼠的巨噬細胞聚落刺激因子 (GM-CSF)的密碼子 (DNA codon)作序列修飾(codon-optmized)，使其mRNA的穩定性提高，藉此提升其蛋白之表達量，之後再以慢病毒載體 (lentivirus)將DNA送入具有HPV致癌基因E6/E7的老鼠腫瘤細胞TC-1中。GM-CSF是重要的造血成長因子，能活化包括T細胞與樹突細胞在內的免疫系統，在此可作為疫苗的佐劑。本篇我們將照過輻射的TC-1/cGM接種在老鼠皮下，並以流氏細胞儀 (flow cytometry)分析疫苗接種後，抗原專一性的T細胞與樹突細胞的免疫反應，再以微型正子照影 (microPET)非侵入性地觀察老鼠體內腫瘤的大小。發現若只打TC-1或TC-1/wtGM (wild-type GM-CSF)作為疫苗，只會增生較少的CD8+ T細胞與樹突細胞，但用TC-1/cGM接種後，可觀察到更強的免疫反應與抗腫瘤效果。

Cervical cancer is the second largest cause of cancer-related death in women Worldwide, which occurs following persistent infection with specific high-risk human papillomaviruses (HPV). Even though the prophylactic vaccines were approved and became available in the market in recent years. There also need therapeutic vaccines to combat high-risk HPV-associated cancers of those had been infected. In order to increase tumor antigen presentation of therapeutic vaccines, we previously modified wild-type GM-CSF gene (wtGM) into codon-optimized GM-CSF (cGM), and then used lentivirus as a vector to deliver GM-CSF gene into a HPV-16 E6/E7 transformed cell line, TC-1. It was verified that TC-1/cGM cells significantly increased steady-state mRNA levels of GM-CSF and further enhanced GM-CSF protein expression. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important hematopoietic growth factor and has profound effects on the functional activities of various leukocytes including T-cells and dendritic cells. In this study, the mice were inoculated with irradiated TC-1/cGM cell-based vaccine, and evaluated its effects on antigen-specific T-cell and dendritic cells (DCs) proliferation and activation by flow cytometric analysis. The antitumor immunity in vivo was monitored using a non-invasive animal position-emission tomography (microPET) imaging. It showed that mice vaccinated with TC-1 or TC-1/wtGM increased a lower level of CD8+ T-cells and DCs immune response. However, administration of TC-1/cGM vaccine revealed more potent immunity to elicit anti-tumor efficacy.

Table of Contents
CHAPTER I Introduction…………..…………………………………. 1
I.1 Cervical cancer and Human Papillomaviruses (HPV)………..… 1
I.2 Prophylactic vaccine of cervical cancer ……………………...… 2
I.3 Therapeutic vaccine of cervical cancer…………………………. 3
I.4 High-dose GM-CSF-secreting tumor cell-based vaccine………...5
CHAPTER II Materials and Methods………………………………….9
II.1 Cell line and mice…………………………………..…………...9
II.2 The differences of GM-CSF secretion among lived and
attenuated cells...……………………………...………….........10
II.3 Mice model of challenge and vaccine immunization………….10
II.4 Measurement of cytokine secretion by ELISA………………...11
II.5 Functional assay of GM-CSF protein……………….................12
II.6 Non-evasive imaging system: MicroPET……………...............13
II.7 Isolation of splenocytes and lymphoid cells………………...…14
II.8 Restimulation of splenocytes and lymphoid cells……………..14
II.9 Flow cytometry analysis………………...……………..………15
II.10 Statistical analysis………………...……………..……………16
CHAPTER III Results………………………………………………...17
III.1 Modification of GM-CSF DNA codon and transgenic tumor
cells.………………...……………..………………..………….17
III.2 GM-CSF expression of living TC-1 and CT-26 cell subsets…..17
III.3 GM-CSF expression of attenuated TC-1 and CT-26 cell
Subsets…………………………………………………………18
III.4 Identical function of GM-CSF secreted by transgenic tumor
cells compared to recombinant protein……………………...…18
III.5 In vivo high-dose GM-CSF secreting tumor vaccine improved
survival of tumor-bearing animals……………………………..19
III.6 High proliferation of cells in total draining lymph nodes of
animals treated with TC-1/wt or TC-1/cGM vaccination……...20
III.7 The efflux of dendritic and macrophage-like cells into
draining lymph nodes was in a GM-CSF dose-dependent
manner…………………………………………………………21
III.8 Entire CD4 T-cell expansion observed in the GM-CSF-
secreting tumor cell-based vaccination……………….………..22
III.9 Activated CD8 T-cell expansion observed in the
GM-CSF-secreting tumor cell-based vaccination……………..23
III.10 The relative cellular frequencies of NK and IKDC subsets
remained unaffected regardless of the presence of GM-CSF….24
III.11 E7-specific T-cell expansion generated by recall response
was restricted in CD8, but not in CD4 T-cells………………..25
III.12 In vitro antigen-specific IFN-γ and IL-4 production by
restmulated splenocytes and DLN cells…………………...…...26
CHAPTER IV Figures………………………………………………...28
IV.1 GM-CSF protein expression level of transgenic TC-1 cell
lysates………………………………………....……………….28
IV.2 Effects of GM-CSF production in gene-transduced tumor
cells of living………………………………....………………..29
IV.3 Effects of GM-CSF production in gene-transduced TC-1
cells after irradiation…………………………………………...30
IV.4 Qualification of GM-CSF produced by gene-transduced TC-1..31
IV.5 In vivo tumor protection following 3 times of vaccination
in C57BL/6 model……………………………………………..32
IV.6 In vivo tumor protection following 3 times of vaccination
in Balb/c model………………………………………………..33
IV.7 Non-invasive micro-positron-emission tomography imaging
(microPET) of in vivo tumor protection experiment……………34
IV.8 Cellularities of immune cells in draining lymph nodes………..35
IV.9 Kinetic induction of macrophage and DCs…………….………36
Kinetic induction of monocytes and DCs…………………… ..36
Kinetic induction of macrophage and DCs in total DLN……...37
Effects of GM-CSF on the cellularities of macrophage or DC
within DLN…………………………………………………….38
IV.10 Kinetic induction of entire CD4 T cells………………………39
IV.11 Kinetic induction of activated CD8 T cells…………………...40
IV.12 Effect of GM-CSF-secreting tumor cell-based vaccine on
NK cell populations…………………………………………..41
IV.13 Recall responses of HPV-specific T cells…………………….42
IV.14 In vitro antigen-specific IFN-γ and IL-4 production after
restimulation………………………………………………….43
CHAPTER V Discussion……………………………………………..44
REFERENCES………………………………………………………….48
Appendix………………...……………………………………………...56

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