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研究生:李昱興
研究生(外文):Yu-Hsin Lee
論文名稱:以胜肽奈米管眼藥水在眼角膜去除後進行抗凋亡基因傳遞研究
論文名稱(外文):Anti-apoptotic Gene Delivery with Peptide Nanotube via Eye Drop Following Corneal Epithelial Debridement
指導教授:廖嘉鴻
指導教授(外文):Jiahorng Liaw
口試委員:林山陽周玉山張淑芬何意
口試委員(外文):Shan-Yang LinShwu-Fen Chang
口試日期:2013-07-11
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:藥學系(碩博士班)
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:57
中文關鍵詞:基因傳遞眼角膜抗凋亡
外文關鍵詞:gene deliverycornealanti-apoptotic
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角膜表皮細胞受傷所引起的角膜基質細胞凋亡,是眼角膜疾病常見致病原因之一。本實驗利用先前研究出的胜肽奈米管,作為基因傳遞地載體,比較質體DNA在胜肽奈米管形成前與形成後加入,兩種不同混合質體DNA與胜肽奈米管的方式,與質體DNA的結合效果,並利用先前建立的角膜去除造成的角膜損傷模式,給予胜肽奈米管混合治療性質體DNA,評估胜肽奈米管混合質體DNA應用於治療角膜損傷的可行性。由原子力顯微鏡與雷射共軛焦顯微鏡可以觀察到所製出的胜肽奈米管長度約在2 ?慆以下,寬度約在200 nm以下。利用DNA會與胜肽奈米管上的酪胺酸(Tyr)產生交互作用,使用螢光光譜觀察胜肽奈米管與質體DNA的交互作用計算出質體DNA在胜肽奈米管形成後加入的結合常數為2.7x108 M-1,而質體DNA在胜肽奈米管形成前加入的結合常數為1.5x107 M-1。觀察兩種混合方式對DNase的安定性發現,則無明顯地差別。並以原子力顯微鏡與雷射共軛焦顯微鏡觀察到胜肽奈米管以及混合質體DNA的胜肽奈米管的型態。利用螢光顯微鏡觀察經由Thioflavin T 染色的胜肽奈米管投予至健康的裸鼠角膜上,可以觀察到胜肽奈米管穿透進入角膜表皮細胞與角膜基質細胞。在眼角膜去除之後的裸鼠眼角膜上,投予標定了Rhodamine的質體DNA與Thioflavin T標定的胜肽奈米管,利用螢光顯微鏡與雷射共軛焦顯微鏡發現胜肽奈米管與質體DNA亦能穿透進入角膜基質細胞。在傷害過後的裸鼠眼角膜上,給予混合胜肽奈米管和兩種不同抗細胞凋亡機轉的質體DNA(pCMV-Bcl-XL-eGFP和caspase 3 silencing shRNA),並進行西方墨點法分析與Caspase 3 酵素活性試驗評估。
Corneal keratocyte apoptosis triggered by cornel debridement is one mechanism of corneal disorders. In this study, the feasibility of peptide nanotubes(PNTs) as carrier of gene delivery between two mixing methods, plamid DNA added before PNTs forming or added after PNTs formed, was compared. Then a model of epithelial injury by epithelial debridement was applied to investigate the feasibility of PNTs as gene delivery carrier on corneal injury. First, the PNTs were found under 2 ?慆 in length, 200 nm in width by atomic force microscope and confocal laser microscope system. Plasmid DNA were observed associated with PNTs by atomic force microscope and confocal laser scanning microscope. The plasmids were added before and after PNT formation and were associated with tyrosine of PNTs with a binding constant of 2.7x108 M-1 and 1.5x107 M-1, respectively. The stability of plasmid DNA with PNTs against the DNase were found no difference between the two formulations. Using thioflavin T stained PNTs on the corneal eye drop delivery, PNTs were observed the distribution on epithelial and stroma region. After corneal debridement, the rhodamine labeled plasmid DNA and thioflavin T stained PNTs were also delivered and could be observed in the stroma of cornea. PNT complexed eyedrop delivery with two anti-apoptotic plasmids (pCMV-Bcl-xL-eGFP and caspase 3 silencing shRNA), were also evaluated by Western blot and caspase 3 activity.
目錄 1
圖表目錄 4
中文摘要 5
Abstract 6
壹、 緒論 7
一、基因傳遞 7
二、載體 9
三、角膜傷害與細胞凋亡 12
貳、 研究目的 17
參、 材料與研究方法 18
一、 實驗試藥 18
二、 儀器與耗材 19
三、 溶液、試劑製備 20
1. Agar plate 20
2. Alkaline Lysis Solution I 21
3. Alkaline Lysis Solution II 21
4. Alkaline Lysis Solution III 21
5. Ethidium Bromide (10mg/ml) 21
6. NaOH (10N) 21
7. 20% SDS 21
8. Tris-HCl (1M; pH 8.8, 7.4, 6.8) 22
9. EDTA (0.5M; pH 8.0) 22
10. 2X SDS gel-loading buffer 22
11. 10X Tris-EDTA buffer (10X TE buffer) 22
12. 1X Tris-EDTA buffer (1X TE buffer) 23
13. 5X Tris-glycine buffer 23
14. 1X Tris-glycine buffer 23
15. 50X TAE buffer 23
16. 0.5X TAE buffer 23
17. Transfer Buffer 23
18. Phosphate Buffer Saline (PBS) 24
19. LB medium 24
20. 10X Tris buffered saline (10X TBS) 24
21. TBST 24
22. Stripping Buffer 24
四、 實驗動物 25
五、 研究方法 25
1. 製備胜肽奈米管 25
2. 製備質體DNA 25
3. TM-Rhodamine標記質體DNA 27
4. 原子力顯微鏡 28
5. 螢光光譜偵測 28
6. 質體DNA混合胜肽奈米管對DNase之安定性試驗 28
7. Zeta potential測量 29
8. Thioflavin T標記胜肽奈米管 29
9. 裸鼠角膜剝除傷害模式 29
10. 活體動物眼藥水投予 30
11. 西方墨點法分析 30
12. Caspase 3 活性分析 31
13. 組織切片分析 31
肆、 實驗結果與討論 32
一、 胜肽奈米管之物化性質 32
1. 胜肽奈米管於顯微鏡下觀察 32
2. Zeta potential測量 34
3. 螢光光譜分析 35
二、 胜肽奈米管混合質體DNA於DNA分解酵素之安定性分析 38
三、 裸鼠投予胜肽奈米管眼藥水之觀察 39
四、 裸鼠角膜傷害與投藥後觀察 41
1. 裸鼠角膜傷害模式 41
2. 螢光標定之質體DNA與胜肽奈米管於顯微鏡觀察 41
3. 西方墨點法分析 44
4. Caspase 3 酵素活性分析 45
伍、 結論 46
陸、 參考文獻 47
Al-Dosari, M. S., & Gao, X. (2009) AAPS J 11, 671-681
Allen, J. E., & McLendon, G. L. (2006) Biochemical and Biophysical Research Communications 349, 1264-1268
Balasubramanian, K., & Burghard, M. (2005) Small 1, 180-192
Binder, C., Read, S. P., Cashman, S. M., & Kumar-Singh, R. (2011) J Gene Med 13, 158-170
Bush, A. (2006) Cystic fibrosis in the 21st century, Karger Medical Scientific
Cavazzana-Calvo, M., Hacein-Bey, S., de Saint Basile, G., Gross, F., Yvon, E., Nusbaum, P., Selz, F., Hue, C., Certain, S., & Casanova, J.-L. (2000) Science 288, 669-672
Chen, C. C., Liu, Y. C., Wu, C. H., Yeh, C. C., Su, M. T., & Wu, Y. C. (2005) Advanced Materials 17, 404-407
Chin, S.-F., Baughman, R. H., Dalton, A. B., Dieckmann, G. R., Draper, R. K., Mikoryak, C., Musselman, I. H., Poenitzsch, V. Z., Xie, H., & Pantano, P. (2007) Experimental Biology and Medicine 232, 1236-1244
Cohen, G. M., Sun, X. M., Fearnhead, H., MacFarlane, M., Brown, D. G., Snowden, R. T., & Dinsdale, D. (1994) Journal of Immunology 153, 507-516
Danhier, F., Ansorena, E., Silva, J. M., Coco, R., Le Breton, A., & Préat, V. (2012) Journal of Controlled Release 161, 505-522
Davis, M. E., Zuckerman, J. E., Choi, C. H., Seligson, D., Tolcher, A., Alabi, C. A., Yen, Y., Heidel, J. D., & Ribas, A. (2010) Nature 464, 1067-1070
Diem, R., Taheri, N., Dietz, G. P. H., Kuhnert, A., Maier, K., Sättler, M. B., Gadjanski, I., Merkler, D., & Bähr, M. (2005) Neurobiology of Disease 20, 218-226
Dua, H. S., King, A. J., & Joseph, A. (2001) British Journal of Ophthalmology 85, 1379-1383
Elsabahy, M., Nazarali, A., & Foldvari, M. (2011) Curr Drug Deliv 8, 235-244
Eriksson, S., Nordén, B., & Takahashi, M. (1993) Journal of Biological Chemistry 268, 1805-1810
Eriksson, S., Norden, B., & Takahashi, M. (1993) Journal of Biological Chemistry 268, 1805-1810
Esquenazi, S., & Mendoza, A. (1999) Journal of refractive surgery (Thorofare, NJ: 1995) 15, 648
Fischer, U., Jänicke, R., & Schulze-Osthoff, K. (2003) Cell Death & Differentiation 10, 76-100
Fuchsluger, T. A., Jurkunas, U., Kazlauskas, A., & Dana, R. (2011) Gene Ther 18, 778-787
Ghadiri, M. R., Granja, J. R., & Buehler, L. K. (1994) Nature 369, 301-304
Ginn, S. L., Alexander, I. E., Edelstein, M. L., Abedi, M. R., & Wixon, J. (2013) J Gene Med 15, 65-77
Goudy, K. S., Wang, B., & Tisch, R. (2008) Clinical immunology 129, 49-57
Han, S., & Roman, J. (2006) Oncogene 25, 4341-4349
Harrison, B. S., & Atala, A. (2007) Biomaterials 28, 344-353
Helena, M. C., Baerveldt, F., Kim, W.-J., & Wilson, S. E. (1998) Investigative Ophthalmology & Visual Science 39, 276-283
Hsieh, W. H., Chang, S. F., Chen, H. M., Chen, J. H., & Liaw, J. (2012) Mol Pharm 9, 1231-1249
Iijima, S. (1991) Nature 354, 56-58
Ji, Z., Zhang, D., Li, L., Shen, X., Deng, X., Dong, L., Wu, M., & Liu, Y. (2009) Nanotechnology 20, 445101
Kaplitt, M. G., Feigin, A., Tang, C., Fitzsimons, H. L., Mattis, P., Lawlor, P. A., Bland, R. J., Young, D., Strybing, K., Eidelberg, D., & During, M. J. (2007) Lancet 369, 2097-2105
Ke, W., Shao, K., Huang, R., Han, L., Liu, Y., Li, J., Kuang, Y., Ye, L., Lou, J., & Jiang, C. (2009) Biomaterials 30, 6976-6985
Kerr, J. F., Wyllie, A. H., & Currie, A. R. (1972) British Journal of Cancer 26, 239-257
Khan, A. S., Fiorotto, M. L., Cummings, K. K., Pope, M. A., Brown, P. A., & Draghia-Akli, R. (2003) American Journal of Physiology - Endocrinology and Metabolism 285, E224-E231
Kilic, E., Dietz, G. P. H., Hermann, D. M., & Bähr, M. (2002) Annals of Neurology 52, 617-622
Kim, R. (2005) Biochemical and Biophysical Research Communications 333, 336-343
Kim, W. J., Rabinowitz, Y. S., Meisler, D. M., & Wilson, S. E. (1999) Experimental eye research 69, 475-481
Klumpp, C., Kostarelos, K., Prato, M., & Bianco, A. (2006) Biochimica et Biophysica Acta (BBA) - Biomembranes 1758, 404-412
Krotz, F., de Wit, C., Sohn, H. Y., Zahler, S., Gloe, T., Pohl, U., & Plank, C. (2003) Molecular Therapy 7, 700-710
Lam, C.-W., James, J. T., McCluskey, R., & Hunter, R. L. (2004) Toxicological Sciences 77, 126-134
Li, Z., Burns, A. R., & Smith, C. W. (2006) Investigative Ophthalmology and Visual Science 47, 1947-1955
Li, Z., & Rana, T. M. (2012) Accounts of chemical research
Liu, W., & Caffrey, M. (2006) Biochemistry 45, 11713-11726
Liu, Z., Winters, M., Holodniy, M., & Dai, H. (2007) Angewandte Chemie International Edition 46, 2023-2027
Lulli, M., Witort, E., Papucci, L., Torre, E., Schipani, C., Bergamini, C., Monte, M. D., & Capaccioli, S. (2012) Investigative Ophthalmology and Visual Science 53, 8295-8302
Marti, G., Ferguson, M., Wang, J., Byrnes, C., Dieb, R., Qaiser, R., Bonde, P., Duncan, M., & Harmon, J. (2004) Gene Ther 11, 1780-1785
Martin, S. J., & Green, D. R. (1995) Cell 82, 349-352
Martin, S. J., Reutelingsperger, C. P. M., McGahon, A. J., Rader, J. A., Van Schie, R. C. A. A., LaFace, D. M., & Green, D. R. (1995) Journal of Experimental Medicine 182, 1545-1556
Mintzer, M. A., & Simanek, E. E. (2008) Chemical Reviews 109, 259-302
Mishra, B., Barik, A., Priyadarsini, K. I., & Mohan, H. (2005) Journal of Chemical Sciences 117, 641-647
Mohan, R. R., Hutcheon, A. E., Choi, R., Hong, J., Lee, J., Mohan, R. R., Ambrósio, R., Zieske, J. D., & Wilson, S. E. (2003) Experimental eye research 76, 71-87
Moret, I., Esteban Peris, J., Guillem, V. M., Benet, M., Revert, F., Dası́, F., Crespo, A., & Aliño, S. F. (2001) Journal of Controlled Release 76, 169-181
Nesic-Taylor, O., Cittelly, D., Ye, Z., Xu, G. Y., Unabia, G., Lee, J. C., Svrakic, N. M., Liu, X. H., Youle, R. J., Wood, T. G., McAdoo, D., Westlund, K. N., Hulsebosch, C. E., & Perez-Polo, J. R. (2005) J Neurosci Res 79, 628-637
Netto, M. V., Mohan, R. R., Sinha, S., Sharma, A., Dupps, W., & Wilson, S. E. (2006) Experimental eye research 82, 788-797
Oliver, L., & Vallette, F. M. (2005) Drug resistance updates 8, 163-170
Pardridge, W. M. (2008) Bioconjugate chemistry 19, 1327-1338
Qazi, Y., Stagg, B., Singh, N., Singh, S., Zhang, X., Luo, L., Simonis, J., Kompella, U. B., & Ambati, B. K. (2012) Investigative Ophthalmology & Visual Science 53, 2837-2844
Ramaesh, T., Ramaesh, K., Leask, R., Springbett, A., Riley, S. C., Dhillon, B., & West, J. D. (2006) Investigative Ophthalmology and Visual Science 47, 1911-1917
Rogers, M.-L., & Rush, R. A. (2012) Journal of Controlled Release 157, 183-189
Saika, S., Shiraishi, A., Liu, C. Y., Funderburgh, J. L., Kao, C. W. C., Converse, R. L., & Kao, W. W. Y. (2000) Journal of Biological Chemistry 275, 2607-2612
Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989) Molecular cloning, Cold spring harbor laboratory press New York
Sedlak, T. W., Oltvai, Z. N., Yang, E., Wang, K., Boise, L. H., Thompson, C. B., & Korsmeyer, S. J. (1995) Proc Natl Acad Sci U S A 92, 7834-7838
Sharma, A., Tandon, A., Tovey, J. C., Gupta, R., Robertson, J. D., Fortune, J. A., Klibanov, A. M., Cowden, J. W., Rieger, F. G., & Mohan, R. R. (2011) Nanomedicine: Nanotechnology, Biology and Medicine 7, 505-513
Sheyn, D., Kimelman-Bleich, N., Pelled, G., Zilberman, Y., Gazit, D., & Gazit, Z. (2008) Gene Ther 15, 257-266
Sokolova, V., & Epple, M. (2008) Angewandte Chemie International Edition 47, 1382-1395
Song, I. K., & Joo, C. K. (2004) Investigative Ophthalmology and Visual Science 45, 423-428
Tong, Y. C., Chang, S. F., Kao, W. W., Liu, C. Y., & Liaw, J. (2010) J Control Release 147, 76-83
Tong, Y. C., Yu, T. Y., Chang, S. F., & Liaw, J. (2012) Mol Pharm 9, 111-120
Tsujimoto, Y., & Shimizu, S. (2000) FEBS Letters 466, 6-10
Wagenknecht, H.-A., Stemp, E. D., & Barton, J. K. (2000) Biochemistry 39, 5483-5491
Walther, W., & Stein, U. (2000) Drugs 60, 249-271
Wendell, D. M., Hemond, B. D., Hogan, N. C., Taberner, A. J., & Hunter, I. W. (2006). The effect of jet parameters on jet injection. Paper presented at the Engineering in Medicine and Biology Society, 2006. EMBS''06. 28th Annual International Conference of the IEEE.
Wilson, S. E., He, Y. G., Weng, J., Li, Q., McDowall, A. W., Vital, M., & Chwang, E. L. (1996) Experimental eye research 62, 325-337
Xiao, J., Wei, X., Wang, Y., & Liu, C. (2009) Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy 74, 977-982
Xing, D., Sun, X., Li, J., Cui, M., Tan-Allen, K., & Bonanno, J. A. (2006) Experimental eye research 82, 780-787
Yang, Z. R., Wang, H. F., Zhao, J., Peng, Y. Y., Wang, J., Guinn, B. A., & Huang, L. Q. (2007) Cancer Gene Therapy 14, 599-615
Yin, W., Cao, G., Johnnides, M. J., Signore, A. P., Luo, Y., Hickey, R. W., & Chen, J. (2006) Neurobiology of Disease 21, 358-371
Zieske, J. D., Guimarães, S. R., & Hutcheon, A. E. K. (2001) Experimental eye research 72, 33-39
Zimmermann, K. C., & Green, D. R. (2001) Journal of allergy and clinical immunology 108, S99-S103
陳正憲. (2012). 溶媒控制及追蹤口服載體基因傳遞之全身性分佈. 碩士, 臺北醫學大學, 台北市. Retrieved from http://ndltd.ncl.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dnclcdr&s=id=%22100TMC05551010%22.&searchmode=basic
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