請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8717
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 何?芳 | |
dc.contributor.author | Yu-Han Wen | en |
dc.contributor.author | 溫裕瀚 | zh_TW |
dc.date.accessioned | 2021-05-20T20:00:08Z | - |
dc.date.available | 2015-03-12 | |
dc.date.available | 2021-05-20T20:00:08Z | - |
dc.date.copyright | 2010-03-12 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-02-09 | |
dc.identifier.citation | 1. Allison, R.R., V.S. Bagnato, R. Cuenca, G.H. Downie, and C.H. Sibata, The future of photodynamic therapy in oncology. Future Oncol, 2006. 2: p. 53-71.
2. Ackroyd, R., C. Kelty, N. Brown, and M. Reed, The history of photodetection and photodynamic therapy. Photochem Photobiol, 2001. 74: p. 656-69. 3. Dolmans, D.E., D. Fukumura, and R.K. Jain, Photodynamic therapy for cancer. Nat Rev Cancer, 2003. 3: p. 380-7. 4. Zhu, T.C. and J.C. Finlay, The role of photodynamic therapy (PDT) physics. Med Phys, 2008. 35: p. 3127-36. 5. Buytaert, E., M. Dewaele, and P. Agostinis, Molecular effectors of multiple cell death pathways initiated by photodynamic therapy. Biochim Biophys Acta, 2007. 1776: p. 86-107. 6. Moor, A.C., Signaling pathways in cell death and survival after photodynamic therapy. J Photochem Photobiol B, 2000. 57: p. 1-13. 7. Calzavara-Pinton, P.G., M. Venturini, and R. Sala, Photodynamic therapy: update 2006. Part 1: Photochemistry and photobiology. J Eur Acad Dermatol Venereol, 2007. 21: p. 293-302. 8. Star, W.M., H.P. Marijnissen, A.E. van den Berg-Blok, J.A. Versteeg, K.A. Franken, and H.S. Reinhold, Destruction of rat mammary tumor and normal tissue microcirculation by hematoporphyrin derivative photoradiation observed in vivo in sandwich observation chambers. Cancer Res, 1986. 46: p. 2532-40. 9. Dolmans, D.E., A. Kadambi, J.S. Hill, C.A. Waters, B.C. Robinson, J.P. Walker, D. Fukumura, and R.K. Jain, Vascular accumulation of a novel photosensitizer, MV6401, causes selective thrombosis in tumor vessels after photodynamic therapy. Cancer Res, 2002. 62: p. 2151-6. 10. Qiang, Y.G., C.M. Yow, and Z. Huang, Combination of photodynamic therapy and immunomodulation: current status and future trends. Med Res Rev, 2008. 28: p. 632-44. 11. Lockshin, R.A. and Z. Zakeri, Programmed cell death and apoptosis: origins of the theory. Nat Rev Mol Cell Biol, 2001. 2: p. 545-50. 12. Edinger, A.L. and C.B. Thompson, Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol, 2004. 16: p. 663-9. 13. Lockshin, R.A. and Z. Zakeri, Apoptosis, autophagy, and more. Int J Biochem Cell Biol, 2004. 36: p. 2405-19. 14. Launay, S., O. Hermine, M. Fontenay, G. Kroemer, E. Solary, and C. Garrido, Vital functions for lethal caspases. Oncogene, 2005. 24: p. 5137-48. 15. Ozben, T., Oxidative stress and apoptosis: impact on cancer therapy. J Pharm Sci, 2007. 96: p. 2181-96. 16. de Bruin, E.C. and J.P. Medema, Apoptosis and non-apoptotic deaths in cancer development and treatment response. Cancer Treat Rev, 2008. 34: p. 737-49. 17. Susnow, N., L. Zeng, D. Margineantu, and D.M. Hockenbery, Bcl-2 family proteins as regulators of oxidative stress. Semin Cancer Biol, 2009. 19: p. 42-9. 18. Szegezdi, E., D.C. Macdonald, T. Ni Chonghaile, S. Gupta, and A. Samali, Bcl-2 family on guard at the ER. Am J Physiol Cell Physiol, 2009. 296: p. C941-53. 19. Ledgerwood, E.C. and I.M. Morison, Targeting the apoptosome for cancer therapy. Clin Cancer Res, 2009. 15: p. 420-4. 20. D'Amelio, M., E. Tino, and F. Cecconi, The apoptosome: emerging insights and new potential targets for drug design. Pharm Res, 2008. 25: p. 740-51. 21. Broughton, B.R., D.C. Reutens, and C.G. Sobey, Apoptotic mechanisms after cerebral ischemia. Stroke, 2009. 40: p. e331-9. 22. Fulda, S., Caspase-8 in cancer biology and therapy. Cancer Lett, 2009. 281: p. 128-33. 23. Martinez-Ruiz, G., V. Maldonado, G. Ceballos-Cancino, J.P. Grajeda, and J. Melendez-Zajgla, Role of Smac/DIABLO in cancer progression. J Exp Clin Cancer Res, 2008. 27: p. 48. 24. Saelens, X., N. Festjens, L. Vande Walle, M. van Gurp, G. van Loo, and P. Vandenabeele, Toxic proteins released from mitochondria in cell death. Oncogene, 2004. 23: p. 2861-74. 25. Jin, S. and E. White, Role of autophagy in cancer: management of metabolic stress. Autophagy, 2007. 3: p. 28-31. 26. Kourtis, N. and N. Tavernarakis, Autophagy and cell death in model organisms. Cell Death Differ, 2009. 16: p. 21-30. 27. Kondo, Y., T. Kanzawa, R. Sawaya, and S. Kondo, The role of autophagy in cancer development and response to therapy. Nat Rev Cancer, 2005. 5: p. 726-34. 28. Martinet, W. and G.R. De Meyer, Autophagy in atherosclerosis: a cell survival and death phenomenon with therapeutic potential. Circ Res, 2009. 104: p. 304-17. 29. Cao, Y. and D.J. Klionsky, Physiological functions of Atg6/Beclin 1: a unique autophagy-related protein. Cell Res, 2007. 17: p. 839-49. 30. Hotchkiss, R.S., A. Strasser, J.E. McDunn, and P.E. Swanson, Cell death. N Engl J Med, 2009. 361: p. 1570-83. 31. Melendez, A. and T.P. Neufeld, The cell biology of autophagy in metazoans: a developing story. Development, 2008. 135: p. 2347-60. 32. MacFarlane, M., Cell death pathways--potential therapeutic targets. Xenobiotica, 2009. 39: p. 616-24. 33. Ranson, M., A. Howell, S. Cheeseman, and J. Margison, Liposomal drug delivery. Cancer Treat Rev, 1996. 22: p. 365-79. 34. Torchilin, V.P., Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov, 2005. 4: p. 145-60. 35. Lasic, D.D., Novel applications of liposomes. Trends Biotechnol, 1998. 16: p. 307-21. 36. Vemuri, S. and C.T. Rhodes, Preparation and characterization of liposomes as therapeutic delivery systems: a review. Pharm Acta Helv, 1995. 70: p. 95-111. 37. Banerjee, R., Liposomes: applications in medicine. J Biomater Appl, 2001. 16: p. 3-21. 38. Jung, S.H., H. Seong, S.H. Cho, K.S. Jeong, and B.C. Shin, Polyethylene glycol-complexed cationic liposome for enhanced cellular uptake and anticancer activity. Int J Pharm, 2009. 382: p. 254-61. 39. Immordino, M.L., F. Dosio, and L. Cattel, Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential. Int J Nanomedicine, 2006. 1: p. 297-315. 40. Derycke, A.S. and P.A. de Witte, Liposomes for photodynamic therapy. Adv Drug Deliv Rev, 2004. 56: p. 17-30. 41. Pagano, R.E. and J.N. Weinstein, Interactions of liposomes with mammalian cells. Annu Rev Biophys Bioeng, 1978. 7: p. 435-68. 42. 黃真宜, 微脂粒包覆Bupivacaine之體外藥物釋離研究, in 藥學研究所. 2001, 國立臺灣大學. p. 113. 43. Agostinis, P., A. Vantieghem, W. Merlevede, and P.A. de Witte, Hypericin in cancer treatment: more light on the way. Int J Biochem Cell Biol, 2002. 34: p. 221-41. 44. Kiesslich, T., B. Krammer, and K. Plaetzer, Cellular mechanisms and prospective applications of hypericin in photodynamic therapy. Curr Med Chem, 2006. 13: p. 2189-204. 45. Davids, L.M., B. Kleemann, D. Kacerovska, K. Pizinger, and S.H. Kidson, Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells. J Photochem Photobiol B, 2008. 91: p. 67-76. 46. Zhao, B., J.J. Yin, P.J. Bilski, C.F. Chignell, J.E. Roberts, and Y.Y. He, Enhanced photodynamic efficacy towards melanoma cells by encapsulation of Pc4 in silica nanoparticles. Toxicol Appl Pharmacol, 2009. 241: p. 163-72. 47. Lam, M., N.L. Oleinick, and A.L. Nieminen, Photodynamic therapy-induced apoptosis in epidermoid carcinoma cells. Reactive oxygen species and mitochondrial inner membrane permeabilization. J Biol Chem, 2001. 276: p. 47379-86. 48. Kessel, D. and M. Castelli, Evidence that bcl-2 is the target of three photosensitizers that induce a rapid apoptotic response. Photochem Photobiol, 2001. 74: p. 318-22. 49. Ichinose, S., J. Usuda, T. Hirata, T. Inoue, K. Ohtani, S. Maehara, M. Kubota, K. Imai, Y. Tsunoda, Y. Kuroiwa, K. Yamada, H. Tsutsui, K. Furukawa, T. Okunaka, N.L. Oleinick, and H. Kato, Lysosomal cathepsin initiates apoptosis, which is regulated by photodamage to Bcl-2 at mitochondria in photodynamic therapy using a novel photosensitizer, ATX-s10 (Na). Int J Oncol, 2006. 29: p. 349-55. 50. Buytaert, E., G. Callewaert, N. Hendrickx, L. Scorrano, D. Hartmann, L. Missiaen, J.R. Vandenheede, I. Heirman, J. Grooten, and P. Agostinis, Role of endoplasmic reticulum depletion and multidomain proapoptotic BAX and BAK proteins in shaping cell death after hypericin-mediated photodynamic therapy. FASEB J, 2006. 20: p. 756-8. 51. Ahn, W.S., S.M. Bae, S.W. Huh, J.M. Lee, S.E. Namkoong, S.J. Han, C.K. Kim, J.K. Kim, and Y.W. Kim, Necrosis-like death with plasma membrane damage against cervical cancer cells by photodynamic therapy. Int J Gynecol Cancer, 2004. 14: p. 475-82. 52. Konan, Y.N., R. Gurny, and E. Allemann, State of the art in the delivery of photosensitizers for photodynamic therapy. J Photochem Photobiol B, 2002. 66: p. 89-106. 53. Siboni, G., H. Weitman, D. Freeman, Y. Mazur, Z. Malik, and B. Ehrenberg, The correlation between hydrophilicity of hypericins and helianthrone: internalization mechanisms, subcellular distribution and photodynamic action in colon carcinoma cells. Photochem Photobiol Sci, 2002. 1: p. 483-91. 54. Fry, D.W., J.C. White, and I.D. Goldman, Rapid separation of low molecular weight solutes from liposomes without dilution. Anal Biochem, 1978. 90: p. 809-15. 55. Boddapati, S.V., G.G. D'Souza, S. Erdogan, V.P. Torchilin, and V. Weissig, Organelle-targeted nanocarriers: specific delivery of liposomal ceramide to mitochondria enhances its cytotoxicity in vitro and in vivo. Nano Lett, 2008. 8: p. 2559-63. 56. Lawrie, A.S., A. Albanyan, R.A. Cardigan, I.J. Mackie, and P. Harrison, Microparticle sizing by dynamic light scattering in fresh-frozen plasma. Vox Sang, 2009. 96: p. 206-12. 57. O'Neal, D., P. Harrip, G. Dragicevic, D. Rae, and J.D. Best, A comparison of LDL size determination using gradient gel electrophoresis and light-scattering methods. J Lipid Res, 1998. 39: p. 2086-90. 58. Galanou, M.C., T.A. Theodossiou, D. Tsiourvas, Z. Sideratou, and C.M. Paleos, Interactive transport, subcellular relocation and enhanced phototoxicity of hypericin encapsulated in guanidinylated liposomes via molecular recognition. Photochem Photobiol, 2008. 84: p. 1073-83. 59. Di Venosa, G., L. Hermida, A. Batlle, H. Fukuda, M.V. Defain, L. Mamone, L. Rodriguez, A. MacRobert, and A. Casas, Characterisation of liposomes containing aminolevulinic acid and derived esters. J Photochem Photobiol B, 2008. 92: p. 1-9. 60. Hayon, T., A. Dvilansky, O. Shpilberg, and I. Nathan, Appraisal of the MTT-based assay as a useful tool for predicting drug chemosensitivity in leukemia. Leuk Lymphoma, 2003. 44: p. 1957-62. 61. Brancaleon, L. and H. Moseley, Laser and non-laser light sources for photodynamic therapy. Lasers Med Sci, 2002. 17: p. 173-86. 62. Usuda, J., S.M. Chiu, E.S. Murphy, M. Lam, A.L. Nieminen, and N.L. Oleinick, Domain-dependent photodamage to Bcl-2. A membrane anchorage region is needed to form the target of phthalocyanine photosensitization. J Biol Chem, 2003. 278: p. 2021-9. 63. Reiners, J.J., Jr., J.A. Caruso, P. Mathieu, B. Chelladurai, X.M. Yin, and D. Kessel, Release of cytochrome c and activation of pro-caspase-9 following lysosomal photodamage involves Bid cleavage. Cell Death Differ, 2002. 9: p. 934-44. 64. Buytaert, E., G. Callewaert, J.R. Vandenheede, and P. Agostinis, Deficiency in apoptotic effectors Bax and Bak reveals an autophagic cell death pathway initiated by photodamage to the endoplasmic reticulum. Autophagy, 2006. 2: p. 238-40. 65. 楊穎奇, 金絲桃素於人類肝癌細胞的攝取路徑與細胞內分佈之探討, in 藥學研究所, 臺灣大學. p. 104. 66. Uzdensky, A., D. Bragin, M. Kolosov, O. Dergacheva, G. Fedorenko, and A. Zhavoronkova, Photodynamic inactivation of isolated crayfish mechanoreceptor neuron: different death modes under different photosensitizer concentrations. Photochem Photobiol, 2002. 76: p. 431-7. 67. Dellinger, M., Apoptosis or necrosis following Photofrin photosensitization: influence of the incubation protocol. Photochem Photobiol, 1996. 64: p. 182-7. 68. Manconi, M., R. Isola, A.M. Falchi, C. Sinico, and A.M. Fadda, Intracellular distribution of fluorescent probes delivered by vesicles of different lipidic composition. Colloids Surf B Biointerfaces, 2007. 57: p. 143-51. 69. Yoshida, T., N. Oide, T. Sakamoto, S. Yotsumoto, Y. Negishi, S. Tsuchiya, and Y. Aramaki, Induction of cancer cell-specific apoptosis by folate-labeled cationic liposomes. J Control Release, 2006. 111: p. 325-32. 70. Molinari, A., M. Colone, A. Calcabrini, A. Stringaro, L. Toccacieli, G. Arancia, S. Mannino, A. Mangiola, G. Maira, C. Bombelli, and G. Mancini, Cationic liposomes, loaded with m-THPC, in photodynamic therapy for malignant glioma. Toxicol In Vitro, 2007. 21: p. 230-4. 71. Wu, J. and M.A. Zern, Modification of liposomes for liver targeting. J Hepatol, 1996. 24: p. 757-63. 72. Yu, H.Y. and C.Y. Lin, Uptake of charged liposomes by the rat liver. J Formos Med Assoc, 1997. 96: p. 409-13. 73. Mahler, S.M., P.A. Wilce, and B.C. Shanley, Studies on regenerating liver and hepatoma plasma membranes--I. Lipid and protein composition. Int J Biochem, 1988. 20: p. 605-11. 74. Mojzisova, H., S. Bonneau, C. Vever-Bizet, and D. Brault, Cellular uptake and subcellular distribution of chlorin e6 as functions of pH and interactions with membranes and lipoproteins. Biochim Biophys Acta, 2007. 1768: p. 2748-56. 75. Kascakova, S., M. Refregiers, D. Jancura, F. Sureau, J.C. Maurizot, and P. Miskovsky, Fluorescence spectroscopic study of hypericin-photosensitized oxidation of low-density lipoproteins. Photochem Photobiol, 2005. 81: p. 1395-403. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8717 | - |
dc.description.abstract | 研究背景
光動力治療(photodynamic therapy;PDT)是一種新興且低侵入性的治療方法,可應用於癌症及其它病症的治療。光動力治療可分為兩個步驟,含光敏感劑經由局部或全身性給藥並給予適當波長的光照。光敏感劑在適當波長的激發後,便會透過能量傳遞產生單態氧及活性氧分子,藉由這些毒性分子的產生,可導致細胞的死亡。但因單態氧的擴散距離短(約20 nm)且持續時間短(約10-6~10-9 s),所以光敏感劑於胞內的分布會決定光照後首要傷害的部位。金絲桃素(hypericin;Hyp)屬於第二代光敏感劑,具有光敏感與螢光的特性,適用於光動力治療及光動力診斷(photodynamic diagnosis),此外,Hyp具單態氧高產率及低暗毒性等優點,故Hyp被認為在光動力治療應用上深具潛力;但因Hyp高脂溶性,在水溶液中易產生聚集(aggregate)的現象,將不利Hyp應用於未來動物或臨床試驗的進行與發展。 研究目的 本研究中,以人類肝癌細胞Hep3B做為體外研究模式的對象。我們利用微脂粒及低密度脂蛋白(low density lipoprotein;LDL)做為Hyp的藥物載體,並且研究各類Hyp製劑在細胞攝取、胞內分布及光動力處理後所引起細胞死亡機制的差異。 研究方法 本研究中以1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)及膽固醇做為微脂粒的主要材料,並添加不同比例的正電荷脂質stearylamine(SA),依SA所佔之莫耳比分為SA0(莫耳比0)、SA0.25(莫耳比0.25)、SA0.5(莫耳比0.5)及SA1.0(莫耳比1.0)等四類liposomal Hyp。製備之liposomal Hyp再以粒徑分析儀及螢光光譜儀分別進行粒徑及包覆率的測定。在微脂粒粒徑穩定性之分析,分別以保存穩定性及光照穩定性檢定,以供確保後續細胞實驗進行期間,微脂粒的粒徑圍完整且均一的。 在細胞實驗中,以流式細胞儀分析Hep3B細胞與各類Hyp製劑於serum-free條件下處理3小時的攝取情形,我們利用螢光影像分析、MTT細胞毒性實驗、流式細胞儀及西方墨點法,觀察各類Hyp製劑於胞內的分布及後續光照處理後引起細胞死亡機制的探討。 研究結果 SA0、SA0.25、SA0.5及SA1.0等各類微脂粒制劑之粒徑依次為183.57 ± 6.80 nm、181.53 ± 10.55 nm、174.87 ± 4.26 nm、177.90 ± 15.50 nm。Hyp包覆率分別為0.094 ± 0.003 μmole Hyp/mg DSPC、0.067 ± 0.003、0.085 ± 0.006及0.078 ± 0.010,其中以SA0及SA0.5的包覆率較高。 在Hyp細胞攝取之研究中,Hep3B細胞在serum-free培養液條件下六類Hyp製劑培養3小時,Hyp攝取量之大小比較為:Hyp>Hyp + 2.5 μg LDL/mL(Hyp-LDL)>SA0>SA0.25 ≒ SA0.5 ≒ SA1.0。在Hep3B和各組Hyp製劑處理3小時後,發現各組製劑均主要以分布在內質網與高基氏體為主,但相較於正電荷liposomal Hyp(SA0.25、SA0.5、SA1.0)等三類製劑組,Hyp組、Hyp + 2.5 μg LDL/mL組及liposomal Hyp(SA0)組中,Hyp之胞內分布較均勻散布於細胞質;而三種正電荷微脂粒組中,Hyp之胞內分布則呈現顆粒狀,且有部份和溶酶體染劑(LysoSensor)產生colocalization的現象。在光動力處理後24小時,發現各組Hyp製劑之細胞毒性大小依序為:Hyp>Hyp-LDL>SA0>SA0.25 ≒SA0.5 ≒ SA1.0,在2 J/cm2光照下,採用各組Hyp的 IC50進行後續的實驗,分別為:Hyp組 28.26 nM、Hyp-LDL組 45.26 nM、SA0組 83.20 nM、SA0.25組 522.89 nM、SA0.5組 551.26 nM、SA1.0組 635.16 nM。各正電荷微脂粒組在細胞攝取、胞內分布及細胞毒性的實驗上,彼此間無顯著差異,故僅以Hyp包覆濃度及包覆率較高的SA0.5製劑進行細胞凋亡機制探討。在Hyp組、Hyp-LDL、SA0及SA0.5各組光動力處理後30分鐘即可發現Hep3B細胞內鈣離子濃度均有明顯上升,各組Hyp製劑和控制組相比均有明顯差異:3.18 ± 0.78(p<0.01)、4.33 ± 0.16(p<0.001)、4.29 ± 0.42(p<0.001)、3.38 ± 0.50(p<0.01)。在光照處理後24小時,Hep3B細胞週期並無停滯現象,但四組之Sub-G1期(細胞凋亡)和控制組相比均有明顯增加(Hyp組(22.36 ± 8.67%;p<0.05)、Hyp-LDL組(18.75± 7.64%;p<0.05)、SA0組(16.41 ± 2.97%;p<0.01)、SA0.5(18.76 ± 3.42%;p<0.01))。為了進一步探究四種Hyp製劑光動力處理後之細胞死亡機制,發現各組均可見endoplasmic reticulum (ER)-stress指標蛋白C/EBP homologous protein(CHOP)及glucose-regulated protein 78(GRP-78)的活化,在細胞凋亡相關蛋白可見到caspase-3及PARP被切割且活化的現象,顯示Hyp光動力處理後將會透過ER-stress引起Hep3B細胞產生細胞凋亡。在細胞自噬指標蛋白LC3I/LC3II(microtubule associated protein light chain 3 I / II)蛋白表現上,並未發現在Hyp光動力處理後LC3II表現有增加的現象,故在本研究處理條件下,Hyp光照處理並未發現Hep3B細胞自噬的現象。 結論 在本研究中發現,各種Hyp製劑於胞內分布主要傾向分布於內質網及高基氏體,各類Hyp製劑光動力處理後將會透過ER-stress(CHOP及GRP-78的活化)引起Hep3B細胞產生細胞凋亡(caspase-3及PARP被切割且活化)。此外,正電荷微脂粒有部份和溶酶體產生colocalization的現象,光動力處理後可能會導致後續細胞死亡機制的不同,但在後續細胞死亡機制的探討上,與其他類製劑組間並無明顯差異。未來如能就Hyp製劑的組織與細胞選擇性及光動力治療效率深入探討,在微脂粒的粒徑、電荷、表面修飾及低密度脂蛋白和Hyp間比例的設計最佳組合,將期能夠在PDT的開發及應用上有所進展。 | zh_TW |
dc.description.abstract | Background
Photodynamic therapy (PDT) is a novel and minimally invasive treatment method for cancer and non-oncological disorders. PDT is a two-step therapeutic technique in which the topical or systemic delivery of photosensitizing drugs is followed by irradiation with appropriate wavelength. After irradiation with suitable wavelength, then activated photosensitizers transfer energy to molecular oxygen, generating singlet oxygen and reactive oxygen species (ROS) that cytotoxicity species could induce cell death. Since the short diffusion distance (20 nm) and half-life (10-6~10-9 s) of singlet oxygen, direct photodamage commonly occurs at the sites where photosensitizer located. Hypericin (Hyp) is classified as second generation photosensitizer, since it has photosensitizing and fluorescence properties suited for photodynamic therapy and photodynamic diagnosis, in addition, Hyp have high quantum yield of photogeneration of singlet oxygen and relatively low dark toxicity, so Hyp is a potential phototherapeutic agent in photodynamic therapy. However, Hyp forms aggregates in aqueous solution, the hydrophobic nature of Hyp limits its ability to study in vivo and clinical trails. Objectives Human hepatoma cell line, Hep3B, was used as in vitro models. We use liposomes and low density lipoprotein (LDL) as Hyp carrier, and study the impacts of different Hyp formulation on cellular uptake, subcellular localization, and Hyp PDT induced cell death mechanisms. Methods In this study, Hyp entrapped liposomes composed of 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) and cholesterol (Chol) with or without addition of different amounts of cationic lipid ,stearylamine (SA), The follow liposomal formulation were tested:SA0 (molar ratio: 0), SA0.25 (molar ratio: 0.25), SA0.5 (molar ratio: 0.5) and SA1.0 (molar ratio: 1.0).The particle size, Zeta potential and Hyp encapsulation of liposomal Hyp were determined by Zetasizer and spectrofluorometer. The liposomes storage and light irradiation stability were also assessed. In vitro study, we examined cellular uptake capacity of Hyp, Hyp-LDL and liposomal Hyp in serum-free medium for 3h by flow cytometry. We used fluorescence image, MTT assay, flow cytometry and western blotting to observe subcellular localization of Hyp and cell death mechanisms induced by PDT treatment. Results Size distribution of liposomes were comparable between four groups for SA0 (183.57 ± 6.80 nm), SA0.25 (181.53 ± 10.55 nm), SA0.5 (174.87 ± 4.26 nm) and SA1.0 (177.90 ± 15.50 nm). In the result of encapsulation efficacy: SA0 (0.094 ± 0.003 μmole Hyp/mg DSPC), SA 0.25 (0.067 ± 0.003), SA 0.5 (0.085 ± 0.006), SA1.0 (0.078 ± 0.010), SA0 and SA0.5 is higher than other groups. In vitro study, Hep3B incubated with different Hyp formulation for 3h in serum-free medium, the extent of uptake is Hyp>Hyp-LDL>SA0>SA0.25≒SA0.5≒SA1.0. Confocal microscopy confirmed the subcellular localization of different Hyp formulation, Hyp mainly colocalized with endoplasmic reticulum (ER) and Golgi apparatus in all Hyp formulation groups. For Hyp, Hyp-LDL and SA0 groups, the fluorescence was found to be diffuse. However, cationic liposomes show granular fluorescence and some colocalize with lysosome specific dye, LysoSensor. The result of Hyp PDT induced cytotoxicity showed at 24h after Hyp PDT treatment: Hyp>Hyp-LDL>SA0>SA0.25 ≒ SA0.5 ≒ SA1.0, then we used IC50 dose of Hyp (Hyp 28.26 nM、Hyp-LDL 45.26 nM、SA0 83.20 nM、SA0.25 522.89 nM、SA0.5 551.26 nM、SA1.0 635.16 nM) at 2 J/cm2 to do following experiment. After Hyp PDT treatment, compared with control, ctyosolic calcium concentration were significantly increase (Hyp-3.18 ± 0.78 (p<0.01)、Hyp-LDL-4.33 ± 0.16 (p<0.001)、SA0-4.29 ± 0.42 (p<0.001)、SA0.5-3.38 ± 0.50 (p<0.01)) and the percentage of Sub-G1 phase(apoptosis)of cell cycle were raise (Hyp (22.36 ± 8.67%;p<0.05)、Hyp-LDL (18.75 ± 7.64%;p<0.05)、SA0 (16.41 ± 2.97%;p<0.01)、SA0.5 (18.76 ± 3.42%;p<0.01) after Hyp PDT treatment, ER-stress marker, C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP-78) were up-regulation, in caspases cascade, caspase-3 and PARP was also cleaved and activated. However, the autophagic marker, LC3I/LC3II (microtubule associated protein light chain 3 I/II), LC3II protein level was not affected by Hyp PDT treatment in Hep3B cell. Conclusion In summary, Hyp mainly localized with ER and Golgi apparatus in all Hyp formulation groups, We found Hyp PDT treatment could induce ER-stress (CHOP and GRP-78 were up-regulation) and led to apoptosis (caspase-3 and PARP was also cleaved and activated) in Hep3B cell. Cationic liposomes showed some colocalization with lysosome. However, there is no different between Hyp formulations in apoptosis signal. In the future, in order to improve tissue selectivity and therapeutic efficacy of Hyp formulation, we would study the size, charge and surface modification of liposomes, and the ratio of concentration of LDL to Hyp, there be a great improvement in the application and development in the field of PDT. | en |
dc.description.provenance | Made available in DSpace on 2021-05-20T20:00:08Z (GMT). No. of bitstreams: 1 ntu-99-R96423018-1.pdf: 3514274 bytes, checksum: 800400005639a59d2b8bc1048da49384 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 中文摘要.................................................I
英文摘要................................................IV 圖目錄..................................................XI 表目錄.................................................XII 英文名詞與簡稱對照表..................................XIII 第壹章 文獻探討..........................................1 1. 光動力治療(Photodynamic Therapy;PDT)................1 1.1 光動力治療發展史.....................................1 1.2 光動力治療之作用機制.................................3 1.3 光動力治療所引起之細胞死亡...........................5 1.3.1 細胞凋亡(Apoptosis)............................5 1.3.2 細胞自噬(Autophagy)............................7 1.3.2 細胞壞死(Necrosis).............................8 2. 微脂粒(Liposome)...................................10 2.1 微脂粒的組成........................................10 2.2 微脂粒的特性與分類..................................10 2.2.1 微脂粒粒徑........................................10 2.2.2 微脂粒表面電荷....................................11 2.2.3 微脂粒於體內穩定性及表面修釋......................11 2.3 微脂粒和細胞的交互作用..............................12 2.3.1 接觸釋放(Contact-release).......................12 2.3.2 吞噬作用(Endocytosis)...........................12 2.3.3 融合(Fusion)....................................12 2.3.4 膜組成交換(Intermembrane Transfer)..............13 2.4轉相溫度(Phase transition temperature).............13 3. 金絲桃素(Hypericin)綜述............................14 3.1 金絲桃素簡介........................................14 3.2 金絲桃素特性........................................14 3.3 金絲桃素光動力處理後引起的細胞死亡..................15 4. 脂蛋白(Lipoprotein)................................17 第貳章 研究動機.........................................18 第參章 實驗材料.........................................19 1. 實驗藥品.............................................19 2. 抗體.................................................21 3. 實驗儀器.............................................22 第肆章 實驗方法.........................................24 1. 微脂粒製備與分析.....................................24 1.1 Liposomal Hypericin製備.............................24 1.2 微型管柱之製備與應用................................24 1.3 微脂粒粒徑及電荷測量................................25 1.4 微脂粒之Hypericin 包覆測量..........................27 1.5 微脂粒之DSPC含量測定................................28 1.6 微脂粒包覆率計算....................................28 1.7 微脂粒穩定性分析....................................29 1.7.1 保存穩定性分析....................................29 1.7.2 光照穩定性分析....................................29 1.8微脂粒轉相溫度測定...................................29 2 細胞實驗..............................................30 2.1 細胞培養............................................30 2.2 細胞計數............................................30 2.3 光照處理............................................30 2.4 細胞毒性分析........................................32 2.5細胞內Hypericin含量測定..............................33 2.6 Hypericin於細胞內之分布.............................34 2.7 細胞內鈣離子濃度測定................................35 2.8 細胞凋亡測定........................................36 2.9 西方墨點法..........................................36 第伍章 實驗結果.........................................40 1. 微脂粒樣品定性及定量.................................40 1.1 微脂粒粒徑、電荷及轉相溫度測量......................40 1.2 微脂粒之Hypericin包覆率分析.........................41 1.3 微脂粒保存穩定性....................................42 1.4 微脂粒光照穩定性測量................................44 2. 細胞對各類Hypericin製劑之攝取分析....................45 3. 各類Hypericin製劑之胞內胞器分布比較..................47 4. 各類Hypericin製劑之細胞毒性分析......................53 5. 各類Hypericin製劑之胞內鈣離子濃度檢測................59 6. 各類Hypericin製劑對細胞週期及細胞凋亡影響............61 7. 各類Hypericin製劑對細胞凋亡及其相關蛋白表現之影響....64 8. 各類Hypericin製劑對細胞自噬相關蛋白表現之影響........67 第陸章 討論.............................................69 第柒章 結論與未來發展方向...............................77 參考文獻................................................78 附錄 附錄一、金斯桃素成份分析................................84 附錄二、低密度脂蛋白產品資訊............................85 附錄三、DSPC產品資訊....................................86 附錄四、Cholesterol產品資訊.............................87 | |
dc.language.iso | zh-TW | |
dc.title | 金絲桃素微脂粒製劑於光動力治療之細胞死亡機制探討 | zh_TW |
dc.title | A Study on the Cell Death Mechanisms of Liposomal Hypericin in Photodynamic Therapy | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 余秀瑛,顧記華 | |
dc.subject.keyword | 光動力治療,金絲桃素,低密度脂蛋白,微脂粒,細胞內分佈,細胞凋亡, | zh_TW |
dc.subject.keyword | photodynamic therapy,hypericin,low density lipoprotein,liposomes,subcellular localization,apoptosis., | en |
dc.relation.page | 87 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2010-02-09 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥學研究所 | zh_TW |
顯示於系所單位: | 藥學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-99-1.pdf | 3.43 MB | Adobe PDF | 檢視/開啟 |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。