Indolicidin之色胺酸殘基對於轉染效率的影響

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：29

、訪客IP：18.191.176.115

姓名

李韋成(Wei-Cheng Li) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

Indolicidin之色胺酸殘基對於轉染效率的影響
(The Effect of Tryptophan Residues on The Transfection Efficiency of Indolicidin)

相關論文

★ 利用穿膜胜肽改善帶正電高分子之轉染效率	★ 利用導電高分子聚吡咯為基材以電刺激促進幹細胞分化
★ 以電刺激增進骨髓基質細胞骨分化之最佳化探討	★ 利用電場控制導電性高分子以進行基因於聚電解質多層膜的組裝
★ 以短鏈胜肽接枝聚乙烯亞胺來進行基因輸送應用之研究	★ 電紡絲製備褐藻酸鈉/聚己內酯之奈米複合纖維進行原位轉染
★ 電場對於複合奈米絲進行原位基因傳送之影響	★ 利用電場調控聚電解質多層膜的釋放以應用於基因輸送
★ 發展載藥電紡聚乳酸/多壁奈米碳管/聚乙二醇纖維	★ 利用寡聚精胺酸促進去氧寡核苷酸輸送
★ 利用聚己內酯/褐藻酸鈉之複合電紡絲擴增癌症幹細胞	★ 以二元體形式之Indolicidin 應用於去氧寡核苷酸之輸送
★ Indolicidin之二聚體形式對輸送去氧寡核?酸的影響	★ 搭建可提供電刺激與機械刺激之生物反應器
★ 硬脂基化的Indolicidin作為傳送質體去氧核酸的非病毒載體	★ 開發促進傷口癒合之複合敷料

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 ( 永不開放)

摘要(中)

在先前的研究當中，我們將Indolicidin(IL) 胜?的N端與C端分別接上半胱胺酸，並分別命名為ILC與CIL。由分子動態模擬發現接枝於聚乙烯亞胺(PEI)的IL是由色胺酸將其疏水片段帶入細胞膜內，來達到基因輸送的效果。為了探討色胺酸對轉染的重要性，因此本研究將ILC序列中第四與第八個色胺酸以及CIL序列中第八與第九個色胺酸改成親水性的甘胺酸，分別命名為ILC48與CIL89，將其以共價接枝或直接摻混結合PEI，以進行質體DNA輸送。由溶血性實驗證實ILC與CIL有強烈的溶血性，反觀改質的ILC48與CIL89則有效降低溶血現象，此趨勢與由MTT assay結果一致，證實減少色胺酸可有助於生物安全性。轉染實驗結果顯示，在接枝的情況下，PEI-ILC及PEI-CIL均可有效地促進轉染，但是PEI-ILC48和PEI-CIL89兩組載體幾乎沒有轉染效率。在直接摻混的實驗方面，由於胜?可藉由其半胱胺酸形成雙硫鍵而成為二元體，而結果顯示，此二元體形式的胜?更能促進轉染效率，其中ILC48與CIL89均有顯著地提昇PEI的轉染效果。除了因為其色胺酸減少所降低的毒性，二元體形式的胜?帶電量較高且分子較長，推測能有助於其與DNA複合的比例，且可提高基因在胞內與載體分離。透過本研究我們了解Indolicidin序列上的色胺酸對於基因輸送的重要性，並有助於設計更安全與高效率的非病毒載體。

摘要(英)

In the previous study, Indolicidin (IL) was added a cystein to its N- and C- terminus, which were denoted as CIL and ILC, respectively. Using molecular dynamics simulations, we found that the grafted IL inserted into the cell membrane mainly related to their tryptophan residues. In order to validate these simulation results, the fourth and eighth tryptophans in the ILC sequence and the eighth and ninth tryptophans in the CIL sequence were replaced with glycine, which were denoted as ILC48 and CIL89, respectively. The hemolysis assay demonstrated that ILC and CIL were highly hemolytic. In contrast, hemolysis caused by ILC48 or CIL89 was low. This trend was consistent to the results of MTT assay, suggesting the reduction of tryptophan should be beneficial to biosaftey. For gene delivery purpose, these peptides were combined with PEI either as conjugates or as mixture to deliver plasmid DNA. In the conjugation form, different from PEI-ILC and PEI-CIL which demonstrated good transfection efficiency, PEI-ILC48 and PEI-CIL89 were unable to transfect cell. Then, we mixed peptides and PEI with DNA as ternary nanocomplexes for transfection. These cysteine-containing peptides can form as dimers through disulfide bond formation. Compared to the monomeric form, dimeric peptides demonstrated superior transfection efficiency. Especially, ILC48 and CIL89 were capable of promoting PEI-mediated gene delivery. We deduced that the dimeric peptide owned more positive charges and larger molecular weights, which increased their complexing ratio to DNA. In addition, their cleavage also facilitated DNA release from peptides intracellularly. Through this comprehensive study, we can determine the importance of tryptophan in peptide for gene delivery. And these results may provide useful information to design a safe and efficient non-viral vector.

關鍵字(中)

★ 聚乙烯亞胺
★ 胜?
★ 二元體
★ 基因傳遞
★ 生物偶聯
★ 色氨酸

關鍵字(英)

★ tryptophan
★ polyethylenimine
★ peptides
★ dimer
★ gene delivery
★ bioconjugation

論文目次

目錄
摘要 I
Abstract III
致謝 IV
目錄 V
圖目錄 IX
表目錄 XI
第一章緒論 1
1-1背景 1
1-2實驗動機 4
第二章文獻回顧 5
2-1基因療法 5
2-2基因載體 6
2-2-1 脂質體複合物 7
2-2-2 正電高分子及其高分子複合物 8
2-3穿膜胜? 12
2-3-1 穿膜胜?的種類與特性 12
2-3-2 Indolicidin 14
2-4利用穿膜胜?與聚乙烯亞胺進行基因輸送 16
2-4-1胜?接枝於聚乙烯亞胺 16
2-4-2三成分摻混對基因的輸送 17
2-5 穿膜胜?的擾膜機制 19
2-6 以二元體形式應用於藥物輸送 22
第三章實驗材料與方法 24
3-1 試藥與原料 24
3-1-1質體DNA 24
3-1-2細胞 25
3-1-3穿膜胜?(Cell-penetrating peptides) 25
3-1-4藥品 26
3-1-5細胞培養用藥 28
3-2 儀器 28
3-3 試藥配製 29
3-4 質體DNA純化 32
3-5 NIH-3T3細胞培養 33
3-6 PEI結合不同種類CPP對轉染效率影響 36
3-6-1細胞轉染 36
3-6-2 ONPG轉染效率分析 41
3-6-3 MTT分析 43
3-7 奈米粒子製備及物理化學性質鑑定 44
3-7-1雷射粒徑分佈儀(dynamic light scattering, DLS)分析 44
3-7-2包覆率分析 44
3-8 Indolicidin之溶血活性 46
3-9 蛋白質膠體電泳(SDS-PAGE)分析 47
3-10載體緩衝能力分析 50
第四章結果與討論 51
4-1胜?接枝PEI應用於藥物輸送 52
4-1-1 PEI衍生物之緩衝能力 52
4-1-2奈米粒子物性鑑定 54
4-1-3粒子包覆率 58
4-1-4溶血性實驗 62
4-1-5 MTT 測試對細胞活性分析 63
4-1-6 PEI結合穿膜胜?對轉染效率之影響 65
4-2胜?與PEI簡單複合應用於藥物輸送 67
4-2-1 SDS-PAGE分析 67
4-2-2奈米粒子物性鑑定 69
4-2-3粒子包覆率 71
4-2-4 Peptide溶血性實驗 74
4-2-5 MTT 測試對細胞活性分析 75
4-2-6 PEI結合穿膜胜?對轉染效率之影響 77
第五章結論 80
參考文獻 82

參考文獻

1. K.M. Boycott, M.R. Vanstone, D.E. Bulman, and A.E. MacKenzie, Rare-disease genetics in the era of next-generation sequencing: discovery to translation. Nature Reviews Genetics, 2013. 14(10): p. 681-691.
2. U. Modlich, J. Bohne, M. Schmidt, C. von Kalle, S. Knoss, A. Schambach, and C. Baum, Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity. Blood, 2006. 108(8): p. 2545-2553.
3. L. Parhamifar, A.K. Larsen, A.C. Hunter, T.L. Andresen, and S.M. Moghimi, Polycation cytotoxicity: a delicate matter for nucleic acid therapy-focus on polyethylenimine. Soft Matter, 2010. 6(17): p. 4001-4009.
4. A. Dehshahri, R.K. Oskuee, W.T. Shier, A. Hatefi, and M. Ramezani, Gene transfer efficiency of high primary amine content, hydrophobic, alkyl-oligoamine derivatives of polyethylenimine. Biomaterials, 2009. 30(25): p. 4187-4194.
5. A. Chugh, F. Eudes, and Y.S. Shim, Cell-Penetrating Peptides: Nanocarrier for Macromolecule Delivery in Living Cells. IUBMB Life, 2010. 62(3): p. 183-193.
6. H. Wang, C.Y. Zhong, J.F. Wu, Y.B. Huang, and C.B. Liu, Enhancement of TAT cell membrane penetration efficiency by dimethyl sulphoxide. Journal of Controlled Release, 2010. 143(1): p. 64-70.
7. A. Baoum, S.X. Xie, A. Fakhari, and C. Berkland, "Soft" Calcium Crosslinks Enable Highly Efficient Gene Transfection Using TAT Peptide. Pharmaceutical Research, 2009. 26(12): p. 2619-2629.
8. A.A. Baoum and C. Berkland, Calcium Condensation of DNA Complexed with Cell-Penetrating Peptides Offers Efficient, Noncytotoxic Gene Delivery. J. Pharm. Sci., 2011. 100(5): p. 1637-1642.
9. S. Yamano, J. Dai, S. Hanatani, K. Haku, T. Yamanaka, M. Ishioka, T. Takayama, C. Yuvienco, S. Khapli, A.M. Moursi, and J.K. Montclare, Long-term efficient gene delivery using polyethylenimine with modified Tat peptide. Biomaterials, 2014. 35(5): p. 1705-1715.
10. H.J. Lee, R. Namgung, W.J. Kim, J.I. Kim, and I.K. Park, Targeted delivery of microRNA-145 to metastatic breast cancer by peptide conjugated branched PEI gene carrier. Macromolecular Research, 2013. 21(11): p. 1201-1209.
11. M.F. Cordeiro, G.S. Schultz, R.R. Ali, S.S. Bhattacharya, and P.T. Khaw, Molecular therapy in ocular wound healing. British Journal of Ophthalmology, 1999. 83(11): p. 1219-1224.
12. P.H. Krebsbach, K. Gu, R.T. Franceschi, and R.B. Rutherford, Gene therapy-directed osteogenesis: BMP-7-transduced human fibroblasts form bone in vivo. Human Gene Therapy, 2000. 11(8): p. 1201-1210.
13. E. Tomlinson and A.P. Rolland, Controllable gene therapy Pharmaceutics of non-viral gene delivery systems (vol 39, pg 357, 1996). Journal of Controlled Release, 1996. 42(3): p. 297-297.
14. G. del Solar, R. Giraldo, M.J. Ruiz-Echevarria, M. Espinosa, and R. Diaz-Orejas, Replication and control of circular bacterial plasmids. Microbiology and Molecular Biology Reviews, 1998. 62(2): p. 434-+.
15. S.C. De Smedt, J. Demeester, and W.E. Hennink, Cationic polymer based gene delivery systems. Pharmaceutical Research, 2000. 17(2): p. 113-126.
16. M.S. Al-Dosari and X. Gao, Nonviral Gene Delivery: Principle, Limitations, and Recent Progress. Aaps Journal, 2009. 11(4): p. 671-681.
17. W. Zauner, S. Brunner, M. Buschle, M. Ogris, and E. Wagner, Differential behaviour of lipid based and polycation based gene transfer systems in transfecting primary human fibroblasts: a potential role of polylysine in nuclear transport. Biochimica Et Biophysica Acta-General Subjects, 1999. 1428(1): p. 57-67.
18. J.F. Xing, L.D. Deng, S.T. Guo, A.J. Dong, and X.J. Liang, Polycationic Nanoparticles as Nonviral Vectors Employed for Gene Therapy in vivo. Mini-Reviews in Medicinal Chemistry, 2010. 10(2): p. 126-137.
19. X.J. Cai, Y.Y. Li, D. Yue, Q.Y. Yi, S. Li, D.L. Shi, and Z.W. Gu, Reversible PEGylation and Schiff-base linked imidazole modification of polylysine for high-performance gene delivery. Journal of Materials Chemistry B, 2015. 3(8): p. 1507-1517.
20. K.L. Douglas, C.A. Piecirillo, and M. Tabrizian, Effects of alginate inclusion on the vector properties of chitosan-based nanoparticles. Journal of Controlled Release, 2006. 115(3): p. 354-361.
21. C. Moreira, H. Oliveira, L.R. Pires, S. Simoes, M.A. Barbosa, and A.P. Pego, Improving chitosan-mediated gene transfer by the introduction of intracellular buffering moieties into the chitosan backbone. Acta Biomaterialia, 2009. 5(8): p. 2995-3006.
22. M. Breunig, U. Lungwitz, R. Liebl, C. Fontanari, J. Klar, A. Kurtz, T. Blunk, and A. Goepferich, Gene delivery with tow molecular weight linear polyethytenimines. Journal of Gene Medicine, 2005. 7(10): p. 1287-1298.
23. S.W. Kim, T. Ogawa, Y. Tabata, and I. Nishimura, Efficacy and cytotoxicity of cationic-agent-mediated nonviral gene transfer into osteoblasts. Journal of Biomedical Materials Research Part A, 2004. 71A(2): p. 308-315.
24. Y.K. Oh, D. Suh, J.M. Kim, H.G. Choi, K. Shin, and J.J. Ko, Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA. Gene Therapy, 2002. 9(23): p. 1627-1632.
25. A. von Harpe, H. Petersen, Y.X. Li, and T. Kissel, Characterization of commercially available and synthesized polyethylenimines for gene delivery. Journal of Controlled Release, 2000. 69(2): p. 309-322.
26. 張萬豐, 探討聚乙烯亞胺轉殖綠螢光蛋白基因進入老鼠胚胎纖維母細胞的最佳條件. 2008. 嘉南藥理科技大學.
27. K. Kilk, S. El-Andaloussi, P. Jarver, A. Meikas, A. Valkna, T. Bartfai, P. Kogerman, M. Metsis, and U. Langel, Evaluation. of transportan 10 in PEI mediated plasmid delivery assay. Journal of Controlled Release, 2005. 103(2): p. 511-523.
28. P. Chollet, M.C. Favrot, A. Hurbin, and J.L. Coll, Side-effects of a systemic injection of linear polyethylenimine-DNA complexes. Journal of Gene Medicine, 2002. 4(1): p. 84-91.
29. D. Fischer, Y.X. Li, B. Ahlemeyer, J. Krieglstein, and T. Kissel, In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials, 2003. 24(7): p. 1121-1131.
30. I.N. Shokolenko, M.F. Alexeyev, S.P. LeDoux, and G.L. Wilson, TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells. DNA Repair, 2005. 4(4): p. 511-518.
31. S. Futaki, Arginine-rich peptides: potential for intracellular delivery of macromolecules and the mystery of the translocation mechanisms. Int. J. Pharm., 2002. 245(1-2): p. 1-7.
32. Y.W. Huang, H.J. Lee, L.M. Tolliver, and R.S. Aronstam, Delivery of Nucleic Acids and Nanomaterials by Cell-Penetrating Peptides: Opportunities and Challenges. Biomed Research International, 2015: p. 16.
33. W.Y. Li, Y.J. Liu, J.W. Du, K.F. Ren, and Y.X. Wang, Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection. Nanoscale, 2015. 7(18): p. 8476-8484.
34. J. Hoyer and I. Neundorf, Peptide Vectors for the Nonviral Delivery of Nucleic Acids. Accounts Chem. Res., 2012. 45(7): p. 1048-1056.
35. J.M. Gump and S.F. Dowdy, TAT transduction: the molecular mechanism and therapeutic prospects. Trends Mol. Med, 2007. 13(10): p. 443-448.
36. B.R. Liu, Y.W. Huang, J.G. Winiarz, H.J. Chiang, and H.J. Lee, Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cell-penetrating peptide through the direct membrane translocation mechanism. Biomaterials, 2011. 32(13): p. 3520-3537.
37. K. Najjar, A. Erazo-Oliveras, D.J. Brock, T.Y. Wang, and J.P. Pellois, An L- to D-Amino Acid Conversion in an Endosomolytic Analog of the Cell- penetrating Peptide TAT Influences Proteolytic Stability, Endocytic Uptake, and Endosomal Escape. Journal of Biological Chemistry, 2017. 292(3): p. 847-861.
38. M. Mae and U. Langel, Cell-penetrating peptides as vectors for peptide, protein and oligonucleotide delivery. Curr. Opin. Pharmacol., 2006. 6(5): p. 509-514.
39. M.E. Selsted, M.J. Novotny, W.L. Morris, Y.Q. Tang, W. Smith, and J.S. Cullor, INDOLICIDIN, A NOVEL BACTERICIDAL TRIDECAPEPTIDE AMIDE FROM NEUTROPHILS. J. Biol. Chem., 1992. 267(7): p. 4292-4295.
40. 蔡秉錩, Indolicidin及其類似物之生物活性與直接穿膜特性. 2012. 國立中央大學.
41. W.W. Hu, Z.W. Lin, R.C. Ruaan, W.Y. Chen, S.L.C. Jin, and Y. Chang, A novel application of indolicidin for gene delivery. International Journal of Pharmaceutics, 2013. 456(2): p. 293-300.
42. H. Li, T.Y. Tsui, and W.X. Ma, Intracellular Delivery of Molecular Cargo Using Cell-Penetrating Peptides and the Combination Strategies. International Journal of Molecular Sciences, 2015. 16(8): p. 19518-19536.
43. E.J. Kwon, S. Liong, and S.H. Pun, A Truncated HGP Peptide Sequence That Retains Endosomolytic Activity and Improves Gene Delivery Efficiencies. Mol. Pharm., 2010. 7(4): p. 1260-1265.
44. Y.M. Lee, D. Lee, J. Kim, H. Park, and W.J. Kim, RPM peptide conjugated bioreducible polyethylenimine targeting invasive colon cancer. Journal of Controlled Release, 2015. 205: p. 172-180.
45. T.T. Zhang, X. Xue, D.L. He, and J.T. Hsieh, A prostate cancer-targeted polyarginine-disulfide linked PEI nanocarrier for delivery of microRNA. Cancer Lett., 2015. 365(2): p. 156-165.
46. C.W. Tsai, Z.W. Lin, W.F. Chang, Y.F. Chen, and W.W. Hu, Development of an indolicidin-derived peptide by reducing membrane perturbation to decrease cytotoxicity and maintain gene delivery ability. Colloids and Surfaces B-Biointerfaces, 2018. 165: p. 18-27.
47. S.J. Lee, S.H. Won, and K.O. Doh, Enhancement of Gene Delivery Using Novel Homodimeric Tat Peptide Formed by Disulfide Bond. Journal of Microbiology and Biotechnology, 2011. 21(8): p. 802-807.
48. J.S. Oh, M. Park, J.S. Kim, and J.H. Jang, Enhanced Cellular Transfection by Ternary Non-Viral Gene Vectors Coupled with Adeno-Associated Virus-Derived Peptides. Macromol. Biosci., 2014. 14(1): p. 121-130.
49. C. Subbalakshmi, V. Krishnakumari, N. Sitaram, and R. Nagaraj, Interaction of indolicidin, a 13-residue peptide rich in tryptophan and proline and its analogues with model membranes. J. Biosci., 1998. 23(1): p. 9-13.
50. V.V. Andrushchenko, M.H. Aarabi, L.T. Nguyen, E.J. Prenner, and H.J. Vogel, Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes. Biochimica Et Biophysica Acta-Biomembranes, 2008. 1778(4): p. 1004-1014.
51. H.A. Rydberg, M. Matson, H.L. Amand, E.K. Esbjorner, and B. Norden, Effects of Tryptophan Content and Backbone Spacing on the Uptake Efficiency of Cell-Penetrating Peptides. Biochemistry, 2012. 51(27): p. 5531-5539.
52. M.L. Jobin, M. Blanchet, S. Henry, S. Chaignepain, C. Manigand, S. Castano, S. Lecomte, F. Burlina, S. Sagan, and I.D. Alves, The role of tryptophans on the cellular uptake and membrane interaction of arginine-rich cell penetrating peptides. Biochimica Et Biophysica Acta-Biomembranes, 2015. 1848(2): p. 593-602.
53. L.C. Hung, I. Jiang, C.J. Chen, J.Y. Lu, Y.F. Hsieh, P.H. Kuo, Y.L. Hung, L.H.C. Wang, M.D.T. Chang, and S.C. Sue, Heparin-Promoted Cellular Uptake of the Cell-Penetrating Glycosaminoglycan Binding Peptide, GBP(ECP), Depends on a Single Tryptophan. Acs Chemical Biology, 2017. 12(2): p. 398-406.
54. 葉喬淳, 以短鏈胜?接枝聚乙烯亞胺來進行基因輸送應用之研究. 2013. 國立中央大學.
55. D.L. McKenzie, K.Y. Kwok, and K.G. Rice, A potent new class of reductively activated peptide gene delivery agents. Journal of Biological Chemistry, 2000. 275(14): p. 9970-9977.
56. D.S. Manickam and D. Oupicky, Multiblock reducible copolypeptides containing histidine-rich and nuclear localization sequences for gene delivery. Bioconjugate Chem., 2006. 17(6): p. 1395-1403.
57. M. Balakirev, G. Schoehn, and J. Chroboczek, Lipoic acid-derived amphiphiles for redox-controlled DNA delivery. Chem. Biol., 2000. 7(10): p. 813-819.
58. M.L. Read, K.H. Bremner, D. Oupicky, N.K. Green, P.F. Searle, and L.W. Seymour, Vectors based on reducible polycations facilitate intracellular release of nucleic acids. Journal of Gene Medicine, 2003. 5(3): p. 232-245.
59. H.L. Amand, B. Norden, and K. Fant, Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation. Biochem. Biophys. Res. Commun., 2012. 418(3): p. 469-474.
60. F. Simeoni, M.C. Morris, F. Heitz, and G. Divita, Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells. Nucleic Acids Research, 2003. 31(11): p. 2717-2724.
61. 黃詩淳, 以二元體形式之Indolicidin應用於去氧寡核?酸之輸送. 2016. 國立中央大學.
62. L.J. Zhang, A. Rozek, and R.E.W. Hancock, Interaction of cationic antimicrobial peptides with model membranes. Journal of Biological Chemistry, 2001. 276(38): p. 35714-35722.
63. G. Grandinetti, N.P. Ingle, and T.M. Reineke, Interaction of Poly(ethylenimine)-DNA Polyplexes with Mitochondria: Implications for a Mechanism of Cytotoxicity. Molecular Pharmaceutics, 2011. 8(5): p. 1709-1719.
64. G. Gasparini, E.K. Bang, G. Molinard, D.V. Tulumello, S. Ward, S.O. Kelley, A. Roux, N. Sakai, and S. Matile, Cellular Uptake of Substrate-Initiated Cell-Penetrating Poly(disulfide)s. Journal of the American Chemical Society, 2014. 136(16): p. 6069-6074.

指導教授

胡威文(Wei-Wen Hu)

審核日期

2018-8-23

推文