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研究生： 王文隆
Wang, Wen-Long
論文名稱： 開發前列腺癌的核酸疫苗及小鼠腫瘤模式
Development of DNA Vaccines and a Murine Tumor Model for Prostate Cancer
指導教授： 吳昭良
Wu, Chao-Liang
學位類別： 碩士
Master
系所名稱： 醫學院 - 生物化學研究所
Department of Biochemistry
論文出版年： 2002
畢業學年度： 90
語文別： 英文
論文頁數： 64
中文關鍵詞： 核酸疫苗 、前列腺癌 、超級抗原 、前列腺特異性抗原
外文關鍵詞： superantigen, DNA vaccine, prostate specific, prostate cancer
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    • 前列腺癌是男人最常見的癌症之一,而且與血液中前列腺特異性抗原(prostate specific antigen, PSA) 的增加有關。PSA是屬於組織特異性的抗原,不論是前列腺的上皮細胞或是前列腺癌細胞都會表現。也由於此原因，前列腺特異性抗原是一個具有潛力的標地物去誘發細胞性免疫反應來對抗前列腺癌細胞。根據文獻報導PSA-3 epitope (前列腺特異性抗原中位在第154-163個胺基酸的重要抗原決定位) 能夠誘發毒殺細胞性T淋巴球 (cytotoxic T lymphocyte, CTL) 的反應。因此本實驗是著重以PSA-3抗原決定位的核酸疫苗來發展前列腺癌疫苗。首先我們利用template-repeat PCR的技術來產生15個PSA-3 epitope，接著將這個片段構築到由大鼠的 b-actin啟動子調控的載體pTCY上。除此之外,某些細菌的蛋白質,如超級抗原 (superantigen) 擁有可以刺激T細胞活化及細胞激素分泌的能力,他們不需要經由MHC class II的呈現,而是直接與T細胞的接受器 (T cell receptor) 作用,進而活化5-20%的T細胞。為了利用超級抗原的生物特性,我們藉著PCR的方法將金黃色葡萄球菌腸毒素A和 B型 (staphylococcal enterotoxin A and B, SEA and SEB) 從S. aureus的染色體DNA選殖出來,構築到pTCY上與15個PSA-3 epitopes形成fusion DNA,所得到質體稱作pTCY-SEA-PSA-3 及pTCY-SEB-PSA-3。將pTCY-SEB-PSA-3以肌肉注射的方式打到小鼠,觀察免疫反應的情況,抗體反應增加由ELISA測得。因為前列腺癌並沒有合適的小鼠腫瘤模式,所以我們必須建立一個小鼠的腫瘤系統,來看我們所設計的疫苗是否有效用。藉由RT-PCR的方式從人類前列腺癌LNCap細胞選殖人類的PSA cDNA,然後構築到由CMV啟動子調控的載體,送到小鼠肝癌細胞ML-1,得到一個穩定的細胞株,稱為PSA/ML-1,此細胞不但會製造PSA,而且會分泌 PSA到細胞外,因此此細胞株可作為小鼠的前列腺癌模式。觀察這些長有PSA/ML-1腫瘤的小鼠,血液中PSA的濃度及腫瘤生長的情形。因為PSA/ML-1與ML-1細胞表現相似量的MHC class I分子,所以可以作為標地細胞偵測CTL針對表現PSA細胞毒殺的反應。更進一步,觀察核酸疫苗在長有PSA/ML-1腫瘤的小鼠是否有抗腫瘤的效用。總而言之,此研究可提供根據PSA以及結合SEB去發展前列腺癌的核酸疫苗。

    Prostate cancer is one of the most common cancer found in man and is associated with increased serum levels of prostate specific antigen (PSA). The expression of PSA is tissue-specific and the majority of prostate cancer and epithelial cells lining the acini and ducts of prostate gland express PSA. For this reason, PSA is a potential target for the induction of cellular immunity against prostate cancer. The PSA-3 (amino acids 154-163) epitope has recently been reported to be capable of eliciting cytotoxic T lymphocyte (CTL) responses. In this study, we aimed to exploit DNA vaccine based on PSA-3 epitope to develop prostate cancer vaccine. The PSA-3 DNA fragment encoding multiple repeats of PSA-3 epitope obtained by template-repeat polymerase chain reaction (TR-PCR) was subcloned into the pTCY vector under the control of the b-actin promoter. In addition, certain bacterial proteins, designated superantigen, possess the ability to stimulate T cell proliferation and cytokine secretion in a process that requires cross-linking of T cell receptor and MHC class II molecules. To exploit the biologic properties of superantigen, we cloned the staphylococcal enterotoxin A and B (SEA and SEB) from S. aureus by PCR. We developed a DNA vaccine construct, pTCY-SEA-PSA-3 and pTCY-SEB-PSA-3, which contained DNA fragments encoding SEA or SEB and 15 copies of PSA-3 epitopes. We investigated the immune response of mice vaccinated intramuscularly with pTCY-SEB-PSA-3. The antibody response measured by ELISA was increased following the vaccination. Because no appropriate murine animal model for prostate cancer was available, we isolated the human PSA cDNA from LNCaP human prostate cancer cells by RT-PCR and cloned into an eukaryotic expression vector controlled by the CMV promoter. The ML-1 mouse hepatoma cell line was transfected with human PSA cDNA and a stable transfectant, designated PSA/ML-1, that expressed PSA and secreted PSA was used as our tumor model. The serum PSA levels and tumor growth were examined in syngeneic Balb/c inoculated with PSA/ML-1 cells. Since PSA/ML-1 cells expressed similar levels of MHC class I molecules compared with the parental ML-1 cells, they can be used as target cells for detecting the activity of CTL response against PSA. Furthermore, the antitumor effect of the DNA vaccine can be tested in mice bearing PSA/ML-1 tumors. In conclusion, this study may pave the way to the development of DNA vaccine based on PSA in combination with SEA or SEB for prostate cancer.

    Chinese abstractⅠ AbstractⅡ Acknowledgement Ⅳ ContentsⅤ Figure contentsⅧ Abbreviation Ⅸ Introduction A.DNA vaccine 1 B.Prostate cancer 2 C.Prostate specific antigen 4 D.Superantigen 5 E.The aim of this study and strategies 6 Materials and methodsA. Materials A.1. The sources of experimental reagents 8 A.2. Plasmids 9 A.3. Oligonucleotides 9 A.4. E coli strains 10 A.5. Cell lines 11 A.6. Experimental model 11 A.7. Cultural media for bacteria 11 A.8. Media for cell culture 12 A.9. Solution 13 A.10. Proteins and antibodies 13 B. Methods B.1. Preparation of plasmid DNA B.1.1. Miniprep plasmid DNA 14 B.1.2. Maxiprep plasmid DNA 14 B.1.3. Determination of DNA amount by spectrophotomete 15 B.2. DNA manipulation B.2.1. Enzyme digestion and gel electrophoresis of DNA 16 B.2.2. Preparation of competent cells 16 B.2.3. Transformation 16 B.3. DNA sequenceing 17 B.4. Cell culture B.4.1. Cell subculture 17 B.4.2. Cell counts using a hemacytometer 17 B.4.3. Transfection B.4.3.1. Transfection by Lipofectin 18 B.4.3.2. Transfection by calcium phosphate 18 B.5. Isolation of genomic DNA from S. aureus 19 B.6. Isolation of cellular mRNA 19 B.7. Reverase transcription (RT) reaction 20 B.8. Polymerase chain reaction (PCR) 21 B.9. Template-repeat PCR (TR-PCR) 25 B.10. Western blot 26 B.11. ELISA 27 B.12. Flow cytometry 27 B.13. DNA preparation and immunization of mice 28 B.14. CTL assay 28 B.15. In vivo tumor growth studies 29 B.16. Collection of sera from tumor-bearing mice 29 B.17. Statistical analysis 29 Results A. Construction of expression vectors carrying PSA-3 repeats alone or fused with SEA or SEB A.1. PSA-3 DNA repeats 30 A.2. Construction of pTCY-SEA-PSA-3 and pTCY-SEB-PSA-3 plasmids 30 A.3. Construction of pTCY-PSA-3 plasmid 31 B.In vitro expression of DNA vaccine constructs in transfected NIH3T3 cells 32 C.Cloning of human PSA cDNA and construction of PSA expression plasmid 32 D.Establishment of a murine tumor cell line expressing human PSA 33 E.FACS analysis of MHC class I molecules in parental ML-1 and PSA/ML-1 cells 33 F.Detection of anti-PSA antibody in mouse sera 34 G.PSA-specific CTL activity 34 H.Growth of ML-1 and PSA/ML-1 tumors 35 Discussion 36 References 41 Figures 48 Appendix 63 Figure contents Fig.1.The strategy of TR-PCR 48 Fig.2.Cloning of SEA and SEB from the genomic DNA of S. aureus 49 Fig.3.Construction of pTCY-SEA-PSA-3 and pTCY-SEB-PSA-3 plasmids 50 Fig.4.Construction of pTCY-PSA-3 plasmid 51 Fig.5.In vitro expression of DNA vaccine constructs in transfected cells 52 Fig.6.Cloning of human PSA cDNA from LNCap humanprostate adenocarcinoma cells 53 Fig.7.Construction of pCMV-PSA plasmid 54 Fig.8.Expression of PSA in the cell lysates of the PSA/ML-1 transfectant 55 Fig.9.Expression of PSA in the culture medium of the PSA/ML-1 transfectant 56 Fig.10.FACS analysis of MHC class I molecules in parental ML-1 and PSA/ML-1 cells 57 Fig.11.Anti-PSA antibody response of mice immunized with DNA vaccine 58 Fig.12.In vitro cytotoxicity assay (Ⅰ) 60 Fig.13.In vitro cytotoxicity assay (Ⅱ) 61 Fig.14.Tumor growth in Balb/c mice inoculated with parental ML-1and PSA/ML-1 cells 62

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