臺灣博碩士論文加值系統

在癌症的基因治療之中，常利用攜帶治療基因的病毒載體作為對抗腫瘤的工具來達成治療癌症的目的。而在本實驗室先前的研究中，以鼠科動物白血病病毒為基礎的可複製型反轉錄病毒（murine leukemia virus-based replicating retroviral vector）作為傳送基因的載體時，被證實是具有高效率的基因傳遞、穩定且持續的基因表達以及高度的腫瘤選擇性。另外，近年來，有一種新的癌症治療策略被發展出來，這個策略主要是在腫瘤細胞中導入自殺基因，讓自殺基因在細胞中表現出特定的酵素，接著加入對細胞本身不具毒性的前藥（prodrug），利用此酵素將前藥轉化為具有極高細胞毒性的化合物來達到毒殺腫瘤的目的。在本篇的研究中，就是要採取結合攜帶自殺基因的病毒載體與酵素-前藥療法的策略來觀察治療肝癌與腦癌的效果。我們首先比較ACE（修改自amphotropic murine leukemia virus, MLV）、GS（修改自Gibbon ape leukemia virus, GaLV）與MSA（含有MLV genome 與GaLV 的envelope基因序列之混合型病毒載體）這三種不同病毒載體的基因傳送效率，在利用流式細胞儀分析被病毒載體感染的細胞的結果中，我們發現這三種病毒載體皆可有效的感染與基因傳送，但其中又以ACE與GS這兩種病毒載體具有較佳的複製傳播效率。接著，觀察攜帶綠色與紅色螢光蛋白基因的ACE與GS病毒載體所感染的腫瘤細胞，我們發現在這些被感染的細胞中皆可表現綠色與紅色的螢光。於是，我們更進一步地在建構病毒載體時，各自將綠色與紅色螢光蛋白基因置換成不同的自殺基因，目的是要測試當加入合適的前藥之後，這些攜帶自殺基因的病毒載體是否能夠因為酵素而轉化成毒性物質，透過毒性物質的釋放而提高腫瘤細胞的毒殺效果，並且也要觀察因為毒性物質的釋放所造成的旁觀者效應（bystander effect）是否有產生。實驗的結果顯示，在Huh-7細胞中，雖然導入單一自殺基因所造成的顯著的細胞毒殺與導入雙重自殺基因的結果並無統計學上的差異，但是在U87細胞中，導入雙重自殺基因所造成的細胞毒殺情形顯著的比導入單一自殺基因的結果要來的多且明顯。因此，利用結合了攜帶自殺基因的病毒載體與酵素-前藥療法作為治療癌症的策略或野i以在癌症的基因治療之中提供了一個有效且新穎的選擇。

Murine leukemia virus (MLV)-based replicating retroviral vectors used for cancer gene therapy are previously proven to be effective, highly stable, tumor-selective, and persistent. Recently, the gene-directed enzyme prodrug therapy (GDEPT) is a widely used approach to increase drug selectivity towards cancer cells. In this investigation, we employed replicating retroviral vectors carrying suicide genes, yeast cytosine deaminase (yCD) and yeast uracil phosphoribosyl transferase (yUPRT), as therapeutic agents for the treatment of liver and brain tumors. First, we compared the gene transfer efficiency of the three replicating retroviral vectors, such as ACE (modified from amphotropic murine leukemia virus, MLV), GS (modified from Gibbon ape leukemia virus, GaLV), and MSA (a hybrid vector containing MLV genome but GaLV’s envelope) vectors, carrying GFP gene as marker, and tried to find out which vector could mediate efficient gene delivery in tumor cells. Our flow-cytometric analysis showed that all of these viral vectors mediated efficient gene delivery in tumor cells, but ACE and GS vectors could mediate better replicative spread. Next, we used ACE and GS vectors carrying GFP and dsRed genes as markers to infect liver and brain tumors, and we found that these marker genes could be simultaneously expressed in transduced cells. In addition to marker genes, we also replaced GFP and dsRed genes in the vectors with suicide genes yCD and yUPRT, respectively, to test cell killing efficiency and to observe bystander effect of resulted vectors upon administration of prodrug 5-fluorocytosine (5-FC). Transduction of dual suicide genes could enhance more tumor cell death after exposure of 5-FC in U87 cells, but there was no significant difference from one suicide gene transferred Huh-7 cells. Therefore, this strategy of combining dual replicating retroviral vectors with dual enzyme/prodrug therapy may serve as attractive tools and provide a new option for cancer gene therapy.

Contents
致謝 i
摘要 ii
Abstract iv
Contents vi
Figure Contents ix
1. Introduction 1
1.1. Tumors 1
1.1.1. Liver tumor 1
1.1.2. Brain tumor 1
1.2. Viral vectors 2
1.3. Replicating retroviral vectors 3
1.4. Receptors for viral entry 4
1.5. Suicide genes 4
1.6. The aims of this study 6
2. Materials and Methods 7
2.1. Materials 7
2.1.1. Plasmids 7
2.1.2. Oligonucleotides 8
2.1.3. Cell lines 9
2.2. Methods 9
2.2.1. Construction of retroviral vectors 9
2.2.2. Transformation and collection of retroviral plasmids 10
2.2.3. Cell lines and cell culture 11
2.2.4. Transfection and retroviral vector production 11
2.2.5. Determination of viral titer 12
2.2.6. Transduction of tumor cells 13
2.2.7. Flow cytometry analysis 13
2.2.8. Confocal microscopic analysis 14
2.2.9. Immunocytochemical analysis 14
2.2.10. Sensitivity of prodrug 5-FC 15
2.2.11. Cell viability and MTS assay 15
2.2.12. Statistical analysis 16
3. Results 17
3.1. Construction of retroviral plasmids 17
3.1.1. Construction of pGS4-yUPRT 17
3.1.2. Construction of pMSA2-dsRed 17
3.2. Replication kinetics of replicating retroviral vectors in tumor cells 17
3.3. Infection with dual replicating retroviral vectors in one tumor cell line 18
3.3.1. Transduction with ACE-GFP and GS4-dsRed in Huh-7 cells 18
3.3.2. Transduction with ACE-GFP and GS4-dsRed in U87 cells 19
3.4. Detection of retroviral-mediated suicide gene transfer in Huh-7 cells by immunocytochemical analysis 20
3.5. Sensitivity of prodrug 5-FC in Huh-7 cells 20
3.6. Tumor cell death with 5-FC administration after suicide gene transfer mediated by replicating retroviral vectors 21
3.6.1. For Huh-7 cells 21
3.6.2. For U87 cells 22
3.7. Tumor cell death with 5-FC administration after mixing suicide gene transferred cells with uninfected cells at various percentages 23
3.7.1. For Huh-7 cells 23
3.7.2. For U87 cells 24
4. Discussion 26
References 31

Figure Contents
Fig. 1. Construction of replicating retroviral vectors 37
Fig. 2. Construction of pGS4-yUPRT 38
Fig. 3. Construction of pMSA2-dsRed 39
Fig. 4. Replication kinetics of replicating retroviral vectors in HCC cell line HA22T/VGH 40
Fig. 5. Replication kinetics of replicating retroviral vectors in HCC cell line HepG2 41
Fig. 6. Replication kinetics of replicating retroviral vectors in HCC cell line Huh-7 42
Fig. 7. Replication kinetics of uninfected HA22T/VGH cells mixed with infected cells 43
Fig. 8. Replication kinetics of uninfected HepG2 cells mixed with infected cells 44
Fig. 9. Replication kinetics of uninfected Huh-7 cells mixed with infected cells 45
Fig. 10. Dual infection with ACE-GFP and GS4-dsRed in Huh-7 cells 46
Fig. 11. Dual infection with ACE-GFP and GS4-dsRed in U87 cells 47
Fig. 12. Detection of infection with ACE-GFP and/or GS4-dsRed by the confocal microscopy in U87 cells 48
Fig. 13. Detection of retroviral envelope SU domain on Huh-7 cells 49
Fig. 14. Detection of suicide gene CD in Huh-7 cells 50
Fig. 15. Sensitivity of prodrug 5-FC on Huh-7 cells 51
Fig. 16. Huh-7 cell death with 5-FC administration after suicide gene transfer mediated by replicating retroviral vectors 52
Fig. 17. U87 cell death with 5-FC administration after suicide gene transfer mediated by replicating retroviral vectors 53
Fig. 18. Huh-7 cell death with 5-FC administration after mixing suicide gene transferred cells with uninfected cells at various percentages. 54
Fig. 19. U87 cell death with 5-FC administration after mixing suicide gene transferred cells with uninfected cells at various percentages 55

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