臺灣博碩士論文加值系統

生物體內最有效針對腫瘤細胞之免疫反應就是細胞毒殺性淋巴球反應(cytotoxic T-lymphocytes, CTLs responce)，而要誘導專一性CTLs的產生必須藉由抗原呈現細胞(antigen presenting cells, APCs)先將特定抗原呈現給CTLs，使CTLs針對帶有此特定抗原之細胞進行毒殺作用。而目前已知最有潛力之APCs即為棘狀細胞(dendritic cells, DCs)。隨著基因工程技術的進步，癌症的基因-免疫治療方法也變得多元化。目前利用基因工程改造過的腫瘤細胞或是受到腫瘤抗原刺激活化的棘狀細胞來進行對癌症的免疫治療構想已被證明可行。在實驗室環境下利用顆粒細胞-巨噬細胞叢聚-刺激因子(Granulocyte-macrophage colony-stimulating factor, GM-CSF)及介白素4號(Interleukin 4, IL-4)，可以刺激由脊髓衍生細胞(bone marrow-derived cells)及腎臟單核細胞(spleen monocytes)轉變為棘狀細胞。而在與腫瘤細胞或腫瘤細胞萃取物共同培養後，棘狀細胞便可成熟為專一性腫瘤抗原呈現細胞。
在本項研究中, 我們的策略是利用基因工程改造過會表現出GM-CSF及IL-4之腫瘤細胞來促使產生成熟之棘狀細胞，並評估此策略對癌症進行免疫基因治療的能力。首先，我們先將小鼠及人類之GM-CSF和IL-4的互補單股去氧核糖核酸(complmentary DNA, cDNA)構築於一重組反轉錄病毒載體(pRufCD)，此載體上已先構築有一自殺基因，即大腸桿菌之胞嘧啶去胺脢(Escherichia coli cytosine deaminase, CD)，有助予以藥物進行正反向篩選。
本實驗是以小鼠之膀胱癌作為腫瘤治療之模式，故選擇小鼠膀胱癌細胞株MBT-2(murine bladder tumor cell lines)作為腫瘤實驗模式細胞。我們利用電穿孔法(electroporation)將帶有小鼠細胞激素cDNA之重組載體送入MBT-2及LL2兩種細胞株。另一方面，我們也將改造過之反轉錄病毒轉染(transfect)進反轉錄病毒包裝細胞(packaging cell lines, CRE and CRIP)，以便於未來可用於將病毒載體送進其他目標細胞。送入重組基因之細胞株，利用自殺基因篩選出後，已利用反轉錄聚合連鎖反應(RT-PCR)證明pmGMCD及pmI4CD兩重組載體已嵌插入腫瘤細胞株(tumor clone cells)之染色體內。爾後利用酵素連結免疫吸附定量法(Enzyme-linked immunosorbent assay, ELISA)檢測腫瘤細胞株分泌細胞激素之能力。而GM-CSF及IL-4是前棘狀細胞(progenitor cells of DCs)轉變為成熟具功能性之棘狀細胞的重要細胞激素，故我們將此會分泌GM-CSF及IL-4之腫瘤細胞株與前棘狀細胞共同培養。我們希望藉此共同培養的方法，使前棘狀細胞受到腫瘤細胞株分泌之細胞激素影響，能夠同時進行捕捉腫瘤抗原及成熟為棘狀細胞，並可將腫瘤抗原呈現給未活化之T細胞(naive T cells)促成生物體內之抗腫瘤免疫反應。我們將利用此策略繼續進行對於癌症之基因治療測試，並嚐試發展具高效能之抗腫瘤疫苗。

Tumor vaccines derived from genetically modified tumor cells or from tumor antigen-pulsed dendritic cells (DCs) show promise in cancer immunotherapy. DCs can be generated from bone marrow-derived cells and splenocytes in the presence of GM-CSF and IL-4. DCs would mature into functional tumor antigen-presenting cells under the exposure to tumor cells or tumor extracts. In this study, the strategies based on DCs combined with genetically engineered tumor cells expressing GM-CSF and IL-4 were exploited to test the feasibility of generation of mature DCs in vitro for tumor immunotherapy. Murine GM-CSF and/or IL-4 cDNA were each cloned into recombinant retroviral vectors (pRufCD), which combine the expression of a suicide gene, E. coli cytosine deaminase (CD), for drug selection. These retroviral vectors were transfected into MBT-2 (murine bladder tumor cell line) and LL2 (murine lewis lung carcinoma cell line). The GM-CSF and IL-4 genes were integrated into the chromosomal DNA of MBT-2 and LL2 cells as confirmed by PCR. The expressions of these two cytokines were also verified by RT-PCR. Furthermore, the GM-CSF protein was detectable in the supernatants of GM-CSF-transfected tumor cells. When splenocytes from naive mice were cultured with MBT-2 cells transduced with GM-CSF gene and those transduced with IL-4 gene, matured DCs were generated, which were characterized for expression of MHC class II antigen, CD11c and CD86. The matured DCs can induce T-cell maturation as well as generate more matured DCs when cocultured with naive splenocytes. We will exploit this DC-based strategy for tumor immunotherapy.

中文摘要…………………………………………………………………I
英文摘要……………………………………………………..…………III
誌謝……………………………………………………………..………IV
目錄…………………………..…………………………………………V
圖目錄………..……………………………………………..…………IX
表目錄………………………..……………………………….……... XII
縮寫與符號………………………………..………………..……….XIII
緒論
A. 顆粒細胞-巨噬細胞叢聚-刺激因子(granulocyte-macrophage colony-stimulating factor, GM-CSF)之生物特性及在免疫-基因治療(immuno-gene therapy)上的角色……………………………...1
B. 介白素4號(Interleukin 4, IL-4) 之生物特性及在免疫-基因治療上的角色……………..…………………………………….……..3
C. 棘狀細胞(dendritic cell)之特性……………………………….…6
D. 大腸桿菌胞嘧啶去胺(Escherichia coli cytosine deaminase, CD) 之特性及生物功能……………………………………………….8
E. 反轉錄病毒載體 (retroviral vector) 之介紹……………………9
F. 膀胱癌模式(Bladder cancer model)之介紹………………….…11
G. 腫瘤的免疫-基因治療(immuno-gene therapy)………….……...12
H. 實驗策略………………………………………………………...14
材料與方法
A. 材料
A.1 實驗試劑來源………………………………………………...15
A.2 質體…………………………………………………………...16
A.3 寡核酸……………………………………………………...16
A.4 菌種…………………………………………………………...17
A.5 細胞株………………………………………………………...17
A.6 實驗動物……………………………………………………...18
A.7 細菌培養液…………………………………………………...18
A.8 細胞培養液……………………………………………….…..18
A.9 溶液…………………………………………………….……..19
B. 方法
B.1 質體置備……………………………………………………...19
B.1.1 小量抽質體的方法 (mini prepare )…………………….19
B.1.2 大量抽質體的方法 (maxi prepare )……………..…..…20
B.1.3 DNA濃度的測量………………………………………..21
B.2 限制切割質DNA………………………………………….21
B.3 回收膠體內之DNA片段……………………………………22
B.4 接合反應 (Ligation)………………………………………….22
B.5 轉型作用 (Transformation)………………………………..…22
B.6 細胞培養………………………………………….…………..23
B.6.1 細胞的次培養………………………………….………..23
B.6.2 細胞的計數………………………………………….…..23
B.7 細胞之質體轉型作用………………………………….……..24
B.8 哺乳動物細胞DNA的抽取……………………….…………25
B.9 聚合連鎖反應……………….…….…….……..…….…….25
B.10 哺乳動物細胞RNA的抽取………………………..………26
B.11 RNA濃度的測量…………………………………..………..27
B.12 反轉錄聚合連鎖反應…………………..……………..27
B.13 酵素連結免疫吸附定量法………………………..………..29
B.14 抗原呈現細胞(APC)與分泌細胞激素之腫瘤細胞共同培養……………………………………………………………..…...30
B.15 流體細胞分析儀……………………………………………31
B.16 磁力細胞挑選回收法………………………………………31
B.17 未曾免疫刺激(naive)之脾臟細胞群受回收共同培養之棘狀細胞刺激增生的T細胞及棘狀細胞表面分子分析…………….32
B.18 細胞毒殺作用之活性分析…………………………………32
結果
A. 構築單獨載有GM-CSF及IL-4基因重組的反轉錄病毒載體phGMCD、phIL4CD、pmGMCD、pmIL4CD及同時載有兩種細胞激素基因之phGMI4CD及pmGMI4CD………………...34
B. 篩選轉染轉錄病毒載體之包裝細胞株………………………..34
C. 定量pmGMCD/MBT-2及pmIL-4/MBT-2細胞株所產生之小鼠GM-CSF及IL-4………………………………………………..35
D. 抗原呈現細胞表面分子分析…………………………………...35
E. 受腫瘤細胞株或棘狀細胞刺激增生之T細胞表面分子分析..36
F. 初次產生之棘狀細胞誘導未曾免疫刺激之脾臟細胞轉變為棘狀細胞之能力分析…………………………………………..….37
G. 細胞毒殺作用…………………………………………..……….37
討論……………………………………………………………………..38
參考文獻…………………………………………………………….….45
圖………………………………………………………………………..51
表………………………………………………………………………..75
附錄……………………………………………………………………..78
自述……………………………………………………………………..82
圖目錄
Fig.1 Construction of phGMCD and phIL4CD plasmids encoding human GM-CSF and IL-4.………………………………………….……51
Fig.2 Construction of pmGMCD and pmIL4CD plasmids encoding human GM-CSF and IL-4……………………………………..…52
Fig.3 Construction of phIL4GMCD plasmids encoding both human GM-CSF and IL-4……………….…………………….………...…….53
Fig.4 Construction of pmIL4GMCD plasmids encoding both mouse GM-CSF and IL-4………………………………………….………….54
Fig.5 Detection of the mouse GM-CSF 419-bp long DNA fragment by PCR in the genomic DNA of tumor cells transduced with the pGMCD vector.………………………………………………..…55
Fig.6 Detection of the mouse IL-4 390-bp long DNA fragment by PCR in the genomic DNA of tumor cells transduced with the pmIL4CD vector.……………………………..……………………………..56
Fig.7 Detection of theCD gene 400-bp long DNA fragment by PCR in the genomic DNA of tumor cells transduced with the pmGMCD or pmIL4CD vector.………………………………………………...57
Fig.8 The expression of mGM-CSF detected by RT-PCR in various tumor clones.……………………………………………………………58
Fig.9 The expression of mIL-4 detected by RT-PCR in various tumor clones.……………………………………………………………59
Fig.10 Detection of mGM-CSF in culture supernatant from mGM-CSF/MBT-2 clones.…………………………………………….60
Fig.11 The mIL-4 standard curve using sandwich ELISA….…………..61
Fig.12.1 FACS analysis of CD11c+MHC-II+ cells in non-adherent splenocytes after coculture with MBT-2 cells transduced with mIL-4 and/or mGM-CSF gene or with recombinant mIL-4 and mGM-CSF.………………………….………………………….62
Fig. 12.2 FACS analysis of CD11c+MHC-II+ cells in non-adherent splenocytes after coculture with MBT-2 cells transduced with mGM-CSF gene or with recombinant mGM-CSF……………..63
Fig.13.1 FACS analysis of CD11c+MHC-II+ cells in non-adherent splenocytes after coculture with MBT-2 cells transduced with mGM-CSF gene or with recombinant mGM-CSF.……….……64
Fig.13.2 FACS analysis of CD86+ cells in non-adherent splenocytes after coculture with MBT-2 cells transduced with mGM-CSF gene or with recombinant mGM-CSF…………………………………..65
Fig.14.1 FACS analysis of CD4+ or CD8+ cells in non-adherent splenocytes after coculture with MBT-2 cells transduced with mIL-4 and/or mGM-CSF gene or with recombinant mIL-4 and mGM-CSF.……………………………………………...……...66
Fig. 14.2 FACS analysis of CD4+ or CD8+ cells in non-adherent splenocytes after coculture with MBT-2 cells transduced with mGM-CSF gene or with recombinant mGM-CSF……………..67
Fig.15.1 FACS analysis of CD4+ or CD8+ cells in non-adherent splenocytes after coculture with DCs obtained from naive splenocytes cocultured previously with MBT-2 cells transduced with mIL-4 and/or mGM-CSF gene or with recombinant mIL-4 and mGM-CSF.…………………………………………………68
Fig.15.2 FACS analysis of CD4+ or CD8+ cells in non-adherent splenocytes after coculture with DCs obtained from naive splenocytes cocultured previously with MBT-2 cells transduced with mGM-CSF gene or with recombinant mGM-CSF………..69
Fig.16.1 FACS analysis of CD11c+ cells in non-adherent splenocytes after coculture with DCs………………………………………………70
Fig.16.2 FACS analysis of CD11c+ cells in non-adherent splenocytes after coculture with DCs……………………………………………….71
Fig.17.1 FACS analysis of CD11c receptor expression in the 1st and 2nd generation of DCs………………………………………………..72
Fig.17.2 FACS analysis of CD11c receptor expression in the 1st and 2nd generation of DCs………………………………………………..73
Fig.18 Cytotoxic T-lymphocyte (CTL) activities by DCs from naive splenocytes cocultured previously with various clones….………74
表目錄
Table 1. Comparisons of the phenotypes of naive splenocytes cocultured with genetic engineered tumor cells or with DCs stimulated with engineered tumor cells…………………………………..75
Table 2. Comparisons of phenotypes of naive splenocytes cocultured with tumor cells or genetic engineered tumor cells.………………..76
Table 3. Comparisons of phenotypes of naive splenocytes cocultured with genetic engineered tumor cells or with DCs stimulated with engineered tumor cells………………………………………..77

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