近年來，免疫療法( immunotherapy)已成癌症治療關注發展的重點之一；本研究希望藉由免疫療法已達治療肝癌( HCC ; hepatocellular carcinoma )。然而，癌細胞具有免疫抑制能力，使免疫療法的成效大為降低。在本文中，吾人利用治療性基因改善免疫療法之治療效率。是否成功運輸這些基因材料至精準的位置並避免與生物體內其他物質產生交互作用為本文之研究之主旨。於本研究中，我們利用已知對HCC具有標靶作用的胜肽修飾雙脂層磷酸鈣奈米載體(Liposome calcium phosphate nanoparticle，LCP-NPs)標靶肝癌細胞。並利用樹枝狀高分子(Dendrimer)來提高基因攜帶率與轉染效果；同時吾人利用奈米粒子將治療性基因運輸至肝癌細胞內，期望藉此增強免疫細胞功能，抑制癌細胞免疫逃脫機制，來達到消滅癌症細胞，治療癌症之效果。

Recently, immunotherapy has become a potential tool for cancer therapy. In this study, we hope to involve immunotherapy in hepatocellular carcinoma (HCC) treatment. However, limitations have been faced by cancer immunotherapy because of immune suppression of cancer cells. In order to resolve these problems, we then use therapeutic gene to improve the efficiency of immunotherapy. This study is focused on developing the novel gene carriers to enhance gene transfection efficient and avoid the non-specific interactions. In our research, we utilized peptide as targeting ligand, by modifying liposome calcium phosphate nanoparticles (LCP-NPs), to specifically target HCC. Meanwhile, the surface-modified dendrimer, which can elevate the transfection efficiency. We delivered the therapeutic gene to increase the efficiency of immunotherapy, which aims to eliminate the cancer cells by immune system. In summary, this targeting LCP-NPs can specifically deliver the therapeutic materials into HCC to enhance the efficiency of immunotherapy.

摘要 i
Abstract ii
致謝 iii
圖目錄 vi
表目錄 vii
縮寫表 viii
第一章、緒論 1
一、研究背景 1
1.1肝癌介紹與形成原因 1
1.2肝癌的治療方法 2
1.3免疫療法 2
二、研究目的 14
第二章、實驗材料與方法 15
2.1 所用材料 15
2.2 細胞培養 16
2.3 動物實驗 16
2.4 製備 LCP-NPs 及 SP94 胜肽表面改質 16
2.5 LCP-NPs 定性分析 17
2.6 SP94-LCP-NPs 細胞攝入與分析表面修飾之樹枝狀高分子運輸 Plasmid DNA至細胞核以及siRNA至細胞質能力 18
2.7 基因轉染率分析 18
2.8 調控基因表現研究 19
2.9 動物腫瘤抑制實驗 20
2.10 數據計算與統計方式 20
第三章、實驗結果與討論 21
3.1 裝載plasmid DNA與siRNA之SP94修飾之磷酸鈣奈米載體之製備與定性分析 21
3.2 觀察搭載表面修飾之樹枝狀高分子的SP94-LCPD-NPs運送plasmid DNA及siRNA至肝癌細胞的攝取表現 23
3.3使用SP94-LCP-NPs運送Luciferase plasmid DNA至肝癌細胞並轉染 Luciferase表現 30
3.4 表面修飾與未修飾之樹枝狀高分子遞送效率比較 33
3.5 SP94-LCPD-NPs運送PD-L1 siRNA調控肝癌細胞PD-L1之表現 36
3.6 製造IL-2細胞激素觀察與分析 38
3.7 小鼠腫瘤抑制實驗與觀察 40
第四章、結果討論與未來計畫 42
第五章、參考文獻 45

1 Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2015. CA: a cancer journal for clinicians 65, 5-29 (2015).
2 Fraumeni, J. F. & Schottenfeld, D. Cancer epidemiology and prevention. (Oxford University Press, 2006).
3 Chan, H. L. et al. Genotype C hepatitis B virus infection is associated with an increased risk of hepatocellular carcinoma. Gut 53, 1494-1498 (2004).
4 El-Serag , H. B. Hepatocellular Carcinoma. New England Journal of Medicine 365, 1118-1127, doi:doi:10.1056/NEJMra1001683 (2011).
5 Farazi, P. A. & DePinho, R. A. Hepatocellular carcinoma pathogenesis: from genes to environment. Nature Reviews Cancer 6, 674-687 (2006).
6 (ASCO), A. S. o. C. O. Liver Cancer - Treatment Options, <http://www.cancer.net/cancer-types/liver-cancer/treatment-options> (2015).
7 Balkhi, M. Y., Ma, Q., Ahmad, S. & Junghans, R. P. T cell exhaustion and Interleukin 2 downregulation. Cytokine 71, 339-347 (2015).
8 Immunology, B. S. f. Introduction of CD8+ T cell, <http://bitesized.immunology.org/cells/cd8-t-cells/> (
9 Health, N. I. o. Immunotherapy, (2015).
10 Oleinika, K., Nibbs, R., Graham, G. & Fraser, A. Suppression, subversion and escape: the role of regulatory T cells in cancer progression. Clinical & Experimental Immunology 171, 36-45 (2013).
11 Gabrilovich, D. I. & Nagaraj, S. Myeloid-derived suppressor cells as regulators of the immune system. Nature Reviews Immunology 9, 162-174 (2009).
12 Mendes, F. et al. The role of immune system exhaustion on cancer cell escape and anti-tumor immune induction after irradiation. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 1865, 168-175 (2016).
13 Ohaegbulam, K. C., Assal, A., Lazar-Molnar, E., Yao, Y. & Zang, X. Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway. Trends in molecular medicine 21, 24-33 (2015).
14 de Coaña, Y. P., Choudhury, A. & Kiessling, R. Checkpoint blockade for cancer therapy: revitalizing a suppressed immune system. Trends in molecular medicine 21, 482-491 (2015).
15 Karachaliou, N. et al. Understanding the function and dysfunction of the immune system in lung cancer: the role of immune checkpoints. Cancer biology & medicine 12, 79 (2015).
16 Blank, C. et al.

PD-L1/B7H-1 Inhibits the Effector Phase of Tumor Rejection by T Cell Receptor (TCR) Transgenic CD8⁺ T Cells

. Cancer Research 64, 1140-1145, doi:10.1158/0008-5472.can-03-3259 (2004).
17 Blank, C., Gajewski, T. F. & Mackensen, A. Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific T cells as a mechanism of immune evasion: implications for tumor immunotherapy. Cancer Immunology, Immunotherapy 54, 307-314, doi:10.1007/s00262-004-0593-x (2005).
18 Blank, C. & Mackensen, A. Contribution of the PD-L1/PD-1 pathway to T-cell exhaustion: an update on implications for chronic infections and tumor evasion. Cancer Immunology, Immunotherapy 56, 739-745, doi:10.1007/s00262-006-0272-1 (2007).
19 Fife, B. T. et al. Interactions between PD-1 and PD-L1 promote tolerance by blocking the TCR–induced stop signal. Nature immunology 10, 1185-1192 (2009).
20 Rivat, C., Santilli, G., Gaspar, H. B. & Thrasher, A. J. Gene Therapy for Primary Immunodeficiencies. Human Gene Therapy 23, 668-675, doi:10.1089/hum.2012.116 (2012).
21 Tripathy, S. K., Black, H. B., Goldwasser, E. & Leiden, J. M. Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. Nat Med 2, 545-550 (1996).
22 Vaheri, A. & Pagano, J. S. Infectious poliovirus RNA: a sensitive method of assay. Virology 27, 434-436 (1965).
23 Sokolova, V. V., Radtke, I., Heumann, R. & Epple, M. Effective transfection of cells with multi-shell calcium phosphate-DNA nanoparticles. Biomaterials 27, 3147-3153 (2006).
24 Niidome, T. & Huang, L. Gene therapy progress and prospects: nonviral vectors. Gene therapy 9, 1647-1652 (2002).
25 Lo, A., Lin, C.-T. & Wu, H.-C. Hepatocellular carcinoma cell-specific peptide ligand for targeted drug delivery. Molecular cancer therapeutics 7, 579-589 (2008).
26 Medina, S. H. et al. Targeting Hepatic Cancer Cells with PEGylated Dendrimers Displaying N-Acetylgalactosamine and SP94 Peptide Ligands. Advanced Healthcare Materials 2, 1337-1350, doi:10.1002/adhm.201200406 (2013).
27 Ashley, C. E. et al. The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers. Nature materials 10, 389-397 (2011).
28 Roy, I., Mitra, S., Maitra, A. & Mozumdar, S. Calcium phosphate nanoparticles as novel non-viral vectors for targeted gene delivery. International Journal of Pharmaceutics 250, 25-33 (2003).
29 Hu, J. et al. A new tool for the transfection of corneal endothelial cells: Calcium phosphate nanoparticles. Acta biomaterialia 8, 1156-1163 (2012).
30 Bisht, S., Bhakta, G., Mitra, S. & Maitra, A. pDNA loaded calcium phosphate nanoparticles: highly efficient non-viral vector for gene delivery. International journal of pharmaceutics 288, 157-168 (2005).
31 Khalil, I. A., Kogure, K., Akita, H. & Harashima, H. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacological reviews 58, 32-45 (2006).
32 Li, J., Chen, Y.-C., Tseng, Y.-C., Mozumdar, S. & Huang, L. Biodegradable calcium phosphate nanoparticle with lipid coating for systemic siRNA delivery. Journal of controlled release 142, 416-421 (2010).
33 Luo, D. & Saltzman, W. M. Synthetic DNA delivery systems. Nature biotechnology 18, 33-37 (2000).
34 Maitra, A. Calcium phosphate nanoparticles: second-generation nonviral vectors in gene therapy. Expert review of molecular diagnostics 5, 893-905 (2005).
35 Yan, J.-Y. et al. Designed nucleus penetrating thymine-capped dendrimers: a potential vehicle for intramuscular gene transfection. Journal of Materials Chemistry B 3, 9060-9066 (2015).
36 Florence, A. T. & Hussain, N. Transcytosis of nanoparticle and dendrimer delivery systems: evolving vistas. Advanced drug delivery reviews 50, S69-S89 (2001).
37 Esfand, R. & Tomalia, D. A. Poly (amidoamine)(PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug discovery today 6, 427-436 (2001).
38 Gillies, E. R. & Frechet, J. M. Dendrimers and dendritic polymers in drug delivery. Drug discovery today 10, 35-43 (2005).
39 Dufès, C., Uchegbu, I. F. & Schätzlein, A. G. Dendrimers in gene delivery. Advanced drug delivery reviews 57, 2177-2202 (2005).
40 Liu, M. & Fréchet, J. M. Designing dendrimers for drug delivery. Pharmaceutical science & technology today 2, 393-401 (1999).
41 Jevprasesphant, R. et al. The influence of surface modification on the cytotoxicity of PAMAM dendrimers. International journal of pharmaceutics 252, 263-266 (2003).
42 Patil, M. L. et al. Surface-modified and internally cationic polyamidoamine dendrimers for efficient siRNA delivery. Bioconjugate chemistry 19, 1396-1403 (2008).
43 Chen, H.-T., Neerman, M. F., Parrish, A. R. & Simanek, E. E. Cytotoxicity, hemolysis, and acute in vivo toxicity of dendrimers based on melamine, candidate vehicles for drug delivery. Journal of the American Chemical Society 126, 10044-10048 (2004).
44 Patri, A. K., Kukowska-Latallo, J. F. & Baker, J. R. Targeted drug delivery with dendrimers: comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex. Advanced drug delivery reviews 57, 2203-2214 (2005).
45 Sadekar, S. & Ghandehari, H. Transepithelial transport and toxicity of PAMAM dendrimers: implications for oral drug delivery. Advanced drug delivery reviews 64, 571-588 (2012).
46 Lee, C. C., MacKay, J. A., Fréchet, J. M. & Szoka, F. C. Designing dendrimers for biological applications. Nature biotechnology 23, 1517-1526 (2005).
47 Devi, G. siRNA-based approaches in cancer therapy. Cancer gene therapy 13, 819-829 (2006).
48 Schiffelers, R. M. et al. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic acids research 32, e149-e149 (2004).
49 Yen, M.-C. et al. A novel cancer therapy by skin delivery of indoleamine 2, 3-dioxygenase siRNA. Clinical Cancer Research 15, 641-649 (2009).
50 Whitehead, K. A., Langer, R. & Anderson, D. G. Knocking down barriers: advances in siRNA delivery. Nature reviews Drug discovery 8, 129-138 (2009).
51 Saffran, D. C. et al. Immunotherapy of established tumors in mice by intratumoral injection of interleukin-2 plasmid DNA: induction of CD8+ T-cell immunity. Cancer gene therapy 5, 321-330 (1997).
52 Stewart, A. K. et al. Adenovector-mediated gene delivery of interleukin-2 in metastatic breast cancer and melanoma: results of a phase 1 clinical trial. Gene therapy 6 (1999).
53 Teo, P. Y. et al. Ovarian Cancer Immunotherapy Using PD‐L1 siRNA Targeted Delivery from Folic Acid‐Functionalized Polyethylenimine: Strategies to Enhance T Cell Killing. Advanced healthcare materials 4, 1180-1189 (2015).
54 Ghafouri-Fard, S. & Ghafouri-Fard, S. siRNA and cancer immunotherapy. (2012).