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研究生:潘曉玲
論文名稱:探討結合IL3及PSA的DNA疫苗對於攝護腺癌治療的可行性
論文名稱(外文):To assess the feasibility of treating prostate cancer with the DNA vaccine contaning PSA and IL-3 genes
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:DNA疫苗PSAIL-3
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摘要
由於腫瘤細胞具有表現它特定腫瘤抗原的特性,此抗原在適當的狀況下會誘發宿主產生特異性的免疫反應,進而殺死腫瘤細胞。本論文探討以攝護腺癌的腫瘤抗原PSA作為DNA疫苗,研究其誘發宿主的免疫反應,以了解其應用於攝護腺癌免疫治療的潛能。不過,DNA疫苗有一個缺點,就是它的效價並不高,所以我們再增加細胞激素IL3(Interleukin 3),一種造血生長因子,由活化的Th或mast 細胞產生,能使幹細胞分化成多種血球細胞,作為輔助劑以增加疫苗的免疫效能。
利用基因重組的技術結合IL-3及PSA,構築出pIRES-IL3-PSA DNA疫苗。在體外實驗中,將pIRES-IL3-PSA DNA轉染到TrampC-1細胞,利用RT-PCR的方式觀察轉染到細胞後的mRNA,發現pIRES-IL3-PSA的IL-3及PSA都有被表現出來,利用ELISA的方式卻只測得IL-3蛋白質的表現。在體內實驗中,利用ELISA的方式測IgG1及IgG2a,發現DNA疫苗並沒有引發老鼠體內的體液性免疫反應,利用CTL分析的方式,雖然有發現標的細胞的數目減少及生長狀況較差,但並沒有引發針對會分泌PSA的Nfsa腫瘤細胞產生毒殺作用的T細胞,而標的細胞死亡的現象可能是NK(nature killing cell)細胞所造成的結果。

Abstract
Tumor cells with the character of expressing tumor specific antigen could be used in immunotherapy. In this study, we explored the possibility of using PSA, a tumor maker for prostate cancer, as DNA vaccine for the treatment of prostate cancer in a mouse model. The disadvantage of DNA vaccine is its efficiency in inducing host immunity. To overcome this obstacle, a cytokine, interleukin-3(IL-3), was used to enhance its efficiency. IL-3 is a haematopoietic growth factor supports the growth and differentiation of pluripoietent stem cells leading to the differentiation of different types of blood cell.
Using gene recombination technique, we constructed a plasmid of pIRES-IL3-PSA for the study of immune efficacy of DNA vaccine. To assess the expression efficiency of this plasmid, this plasmid was transfected into Tramp-C1 cells. The mRNA expression of this plasmid in TRAMP-C1-IRES-IL3-PSA cell was examined by RT-PCR. The results demonstated that IL-3 and PSA mRNAs could be expressed in Trammp-C1-IRES-IL3-PSA cells. To further evaluate the production of these proteins in this cell, ELISA assay was performed. The results demonstrated that these cells could express IL-3 protein, but not detectable PSA protein.
Following two doses of intra-muscular injection of this plasmid, serum IgG1 and IgG2a levels were measured to evaluate the immune responses. ELISA result showed that the DNA vaccine couldn’t induce humoral immunity in mice. To further evaluate the cellular immune response, CTL assay was performed. CTL assay showed that DNA vaccine could induce CTL response against Nfsa-PSA cells. This indicated that DNA vaccine could induce cell-mediate immunity in mice. However, this response occurred in both C3H/HeN and C57BL/6J mice indicated that the observed response is likely due to the non-specific killing of NK cells.

目錄
英文摘要 I
中文摘要 III
誌謝 V
目錄 VI
第一章、 緒論
1.1攝護腺癌及傳統治療方式 1
1.2免疫治療 3
1.3免疫系統 3
1.4 DNA疫苗 4
1.5細胞激素(Cytokine) 6
1.6攝護腺特異性抗原(Prostate-specific-antigen) 7
1.7介白素三號(Interluekin-3) 8
1.8實驗目的 9
第二章、 材料與方法
2.1細胞培養 10
2.2質體的構築
2.2.1.DNA製備
2.2.1.1.聚合酵素連鎖反應(Polymerase Chain Reaction) 10
2.2.1.2.電泳分析 11
2.2.1.3.限制酵素切割反應(Restriction Enzyme Digestion)11
2.2.1.4.去磷酸化(Dephosphorylation) 12
2.2.1.5. DNA接合(Ligation) 13
2.2.1.6.勝任細胞製備(Competent Cell) 13
2.2.1.7.細胞熱休克轉殖法(Heat-Shock Transformation) 14
2.2.1.8.微量DNA放大 15
2.2.1.9.大量DNA放大 15
2.3酵素連結免疫吸附法(Enzyme-linked Immuno-sorption Assay)
17
2.4動物實驗
2.4.1.實驗動物 18
2.4.2.大腿肌肉注射 18
2.4.3.血清樣品取得 18
2.4.4.T淋巴球取得 18
2.5毒殺型T淋巴球分析(cytotoxic T lymphocyte assay) 20
2.6細胞的轉染(Transfection) 20
2.7 RNA的抽取(Total RNA Isolation) 21
2.8 RT-PCA
2.8.1反轉錄作用(Reverse Transcription) 22
2.8.2聚合酵素連鎖反應(PCR) 22
2.9細胞對抗生素藥物敏感度測試 23
第三章、 實驗結果
3.1構築結合IL-3及PSA的DNA疫苗 24
3.2 pIRES-IL3-PSA基因在轉染細胞中的表現
3.2.1利用liposome將pIRES-IL3-PSA DNA送至細胞中 26
3.2.2在轉染細胞中pIRES-IL3-PSA DNA被轉錄為mRNA的情形 27
3.2.3在轉染細胞中pIRES-IL3-PSA DNA被轉譯為蛋白質的情形 28
3.3以pIRES-IL3-PSA為DNA疫苗,在C3H/HeN及C57/B6J兩種不同品系老鼠中的免疫反應
3.3.1血清中免疫球蛋白IgG1的ELISA分析 28
3.3.2血清中免疫球蛋白IgG2a的ELISA分析 30
3.3.3血清中IL-3及PSA的ELISA分析 31
3.3.4毒殺型T細胞的作用效果 31
第四章、 討論 33
第五章、 參考文獻 39
圖表 41
附錄 60

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