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研究生:伍凰碧
論文名稱:評估結合PSA攝護腺特異性抗原與細胞激素GM-CSF之DNA疫苗在治療攝護腺癌的可行性
指導教授:江啟勳
學位類別:碩士
校院名稱:國立清華大學
系所名稱:原子科學系
學門:工程學門
學類:核子工程學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:攝護腺特異性抗原攝護腺癌基因疫苗
外文關鍵詞:PSAGM-CSFprostate cancerDNA vaccine
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摘要
本實驗的目的評估結合PSA攝護腺特異性抗原與細胞激素GM-CSF之DNA疫苗在治療攝護腺癌的可行性。首先建立一個可同時表現人類PSA基因與老鼠GMCSF基因的DNA疫苗,再來我們將此質體DNA注射到C57/B6J與C3H/HeN兩種品系的老鼠體內比較不同的品系引起免疫反應,並與單獨表現人類PSA基因的DNA疫苗所引起的免疫反應作比較來評估。
結果發現此DNA疫苗在兩種品系老鼠體內並未引起體液性免疫反應,在血清中測得因肌肉注射引起的IgG1與IgG2a並沒有顯著差異。以西方墨點法也無法從血清中測得anti-PSA抗體的存在,然而從注射過疫苗的老鼠體內取出的脾臟純化出T細胞所做的毒殺型T細胞試驗可看出毒殺Nfsa-PSA的效果。但是兩種品系的老鼠皆可觀察到相同的毒殺效果,因此可能為殺手細胞所引起的非專一型的毒殺。
此構築的PSA之DNA疫苗在引起特異性免疫作用方面為失敗的,原因是我們只能以RT-PCR證實NFsa-PSA在mRNA有表現PSA,但不論在上清液若細胞溶解後的蛋白質中皆無法以ELISA測到。皆無法測得其存在。這可能是因我們所構築的PSA中的點突變或缺少PSA基因非轉譯的區域所造成。要解決此一問題,得以序列完全相同的PSA基因才行。另外使用PSA的轉殖基因鼠或使用老鼠本身的攝護腺抗原probasin與GM-CSF結合當作疫苗為建議改善的方法。

Abstract
The aim of this study is to assess the feasibility of treating prostate cancer with the DNA vaccine containing PSA and GM-CSF genes. First, we cloned a DNA plasmid to express the gene of human PSA and the gene of mice GM-CSF simultaneously. Second, we injected this plasmid into C57BL/6J and C3H/HeN mice to evaluate the immunity of different strains of mice. The effect of GM-CSF on DNA vaccine was evaluated by comparing its immunity with the immunity generated by PSA alone.
The results of this study showed that constructed plasmid could not generate humoral immunity against PSA in both strains of mice. There was not difference in IgG1 or IgG2a level following intramuscular DNA vaccine. Western blot was unable to detect the anti-PSA antibody in the serum. However, the CTL assay showed the cytotoxicity of spleen cells from immuned-mice against NFsa-PSA cells. The CTL responses were observed in both strains of mice, which indicated that this response is likely due to the non-specific killing of NK-cells.
The failure of the generation of specific immunity following DNA vaccine could be the results of PSA cloning. Because we found that Nfsa-PSA cells could only express mRNA of PSA as assessed by RT-PCR. We were unable to detect hPSA protein in both the supernatant and cell lysis by ELISA assay. We are currently unaware whether this effect is due to the point mutations of PSA that we cloned or the lack of non-translated regions of PSA gene. A clone of without mutation could be one way to answer this question. Beside, to improve this model, the use of PSA transgenic mice or probasin antigen, which is the prostate specific-antigen of the mouse, is suggested.

目錄
中文摘要……………………………………………………….I
英文摘要……………………………………………………...III
誌謝…………………………………………………………....V
目錄…………………………………………………………...VI
第一章、 前言
1.1 攝護腺癌……………………………………………………1
1.2 癌症免疫治療………………………………………………2
1.2.1 DNA疫苗…………………….…………………....4
1.3 攝護腺癌發展中的免疫治療……………………………....6
1.4 攝護腺特異性抗原…………………….…………………...7
1.5 顆粒球巨噬細胞群落刺激因子….………………………...9
1.6 研究目的與內容…………………………………………..11
第二章、 材料與方法
2.1 細胞培養………………………………..………… ……...12
2.2 質體的構築.…………………………………………….…12
2.2.1 勝任細胞(Competent Cell)的製備…….….….12
2.2.2 細菌熱休克轉殖法(Heat-Shock Transformation)……………………………….....13
2.2.3 質體的製備DNA……..…………………….……...14
2.2.3.1 微量DNA製備(miniprep)………..………...14
2.2.3.2 中量製備DNA(midiprep).…….…………...14
2.2.3.3 大量製備DNA(maxiprep)……………….15
2.2.4 RNA的純化(Total RNA Isolation)……….…….16
2.2.5 RT-PCR…………………….………………….…17
2.2.5.1 反轉錄作用 (Reverse Transcription).………17
2.2.5.2 聚合酵素連鎖反應……………………...…..18
2.2.6 洋菜膠電泳……………………………….……...18
2.2.7 DNA片段的分離(Gel Extraction)……….…..19
2.2.8 限制酵素切割反應…………….………….……..19
2.2.9 DNA的純化(Purification).……………………...20
2.2.10 DNA片段的去磷酸化(de-phosphoylation).……20
2.2.11 DNA的接和(Ligation)………………..………...21
2.2.12 聚合酵素連鎖反應…………………………..…..21
2.3 在轉染細胞中的表現…………………………………………22
2.3.1 細胞對Puromycin的敏感性試驗I…………...…22
2.3.2 細胞存活試驗 (MTT assay)………………...…..23
2.3.3 細胞對Puromycin的敏感性試驗II…………......23
2.3.4 細胞的轉染 (Transfection)…………………..….24
2.3.5 酵素連結免疫吸附法……………………………24
2.3.5.1 GMCSF ELISA……………………………...24
2.2.5.2 PSA ELISA kit…………………………...….26
2.4 在動物體內的表現………………………………………...….26
2.4.1 實驗動物…………………………………………26
2.4.2 肌肉注射…………………………………………26
2.4.3 血清的抽取………………………………………27
2.4.4 酵素連結免疫吸附法……………………………27
2.4.4.1 IgG1、IgG2a ELISA……………..…………27
2.4.4.2 Anti-PSA ELISA ……………………………28
2.4.5 西方墨點法(Western blot)………………………29
2.4.5.1 細胞的溶解………………………………….29
2.4.5.2 製備電泳膠………………………………….29
2.4.5.3 免疫轉印…………………….………………30
2.4.6 T細胞的純化…………………………………….30
2.4.6.1 備製尼龍管柱(nylon column)……………30
2.4.6.2 細胞處理…………………………………….31
2.4.6.3 漲破血球…………………………………….31
2.4.6.4 純化T細胞………………………………….31
2.4.7 毒殺型T細胞試驗(CTL assay)………………32
第三章、 結果
3.1 構築IRES-PSA-GMCSF之DNA疫苗……………………….33
3.1.1 構築IRES-PSA-GMCSF之質體DNA…………33
3.2 注射IRES-PSA-GMCSF與IRESp-PSA到C57/B6J 和C3H/HeN兩種品系中,並測試所引起的免疫反應…………35
3.2.1 施打DNA疫苗並採取血清…………………….35
3.2.2 血清中IgG1之ELISA結果……………………36
3.2.3 血清中IgG2a之ELISA結果…………………….37
3.2.4 血清中PSA之ELISA結果…………………….39
3.2.5 血清中GMCSF之ELISA結果………………….39
3.2.6 西方墨點法測血清中Anti-PSA抗體之結果…...39
3.3 建立 Nfsa-PSA細胞…………………………………………..40
3.3.1 細胞對Puromycin的敏感性試驗I……………...41
3.3.2 細胞對Puromycin的敏感性試驗II…………….41
3.3.3 轉染細胞(Nfsa-PSA)中人類PSA的mRNA表現………………………………………………….41
3.3.4 在轉染細胞中(Nfsa-PSA)中人類PSA蛋白質的表現……………………………………………….42
3.4 注射DNA疫苗的老鼠之毒殺型T細胞試驗(CTL assay)果………………………………………………………44
第四章、討論…………………………………………………………46
第五章、參考文獻……………………………………………54
圖表……………………………………………………………58
附錄一…………………………………………………………78
附錄二…………………………………………………………79

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