研究生: |
黃景鴻 Huang, Ching-Hong |
---|---|
論文名稱: |
以材料為核心探索 : 透過抑制聚集以穩定人類降鈣素 A material-centric exploration : Stabilizing human calcitonin by inhibiting aggregation |
指導教授: |
杜玲嫻
Tu, Ling-Hsien |
口試委員: |
杜玲嫻
Tu, Ling-Hsien 李以仁 Lee, I-Ren 葉伊純 Yeh, Yi-Cheun |
口試日期: | 2024/07/30 |
學位類別: |
碩士 Master |
系所名稱: |
化學系 Department of Chemistry |
論文出版年: | 2024 |
畢業學年度: | 112 |
語文別: | 中文 |
論文頁數: | 73 |
中文關鍵詞: | 人類降鈣素 、類澱粉蛋白聚集 、史托伯法 |
英文關鍵詞: | human calcitonin, amyloid aggregation, Stöber process |
研究方法: | 實驗設計法 |
DOI URL: | http://doi.org/10.6345/NTNU202401606 |
論文種類: | 學術論文 |
相關次數: | 點閱:59 下載:0 |
分享至: |
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
人類降鈣素(human calcitonin, hCT)是一種由32個胺基酸所組成的荷爾蒙胜肽。在生物體內扮演調節血鈣水平的重要角色,可抑制破骨細胞的活動,因此常被作為藥物使用來治療骨骼相關疾病,例如: 佩吉特氏病和骨質疏鬆症等。由於它高聚集傾向的特性,導致其藥物效果受到一定限制,我們的研究目標在於開發新的材料用於穩定該胜肽。根據先前實驗室的研究發現,醣類分子對抑制hCT的聚集具有潛在能力,但需要在高濃度下才能發揮聚集抑制的作用。在這項研究中,我們利用史托伯法 (Stöber process )製備溶膠,並引入額外的葡萄糖,希望能有效地串聯溶液中的葡萄糖分子,藉此降低葡萄糖的濃度或提升抑制聚集的效果。在本研究中,我們參考了相關文獻中的製備條件,以獲得穩定的樣品溶液,之後再進行製備條件的優化。當製備出的材料穩定度提升後,我們才開始調整添加的葡萄糖濃度。首先,透過凝膠滲透色譜(Gel permeation chromatography, GPC)判斷材料的交聯程度,確保其交聯程度相似後,再以傅立葉轉換紅外光譜(Fourier-transform infrared spectroscopy, FTIR)確認葡萄糖是否交聯於材料中。接著,我們利用X射線光電子能譜(X-ray photoelectron spectroscopy, XPS)分析材料之元素比例 ,推算出材料中交聯的葡萄糖量。在硫磺素T(Thioflavin T, ThT)動力學實驗中,我們觀察到材料通過減少共同培育下的hCT纖維數量方式達到抑制hCT聚集的效果,該結果也透過穿透式電子顯微鏡(Transmission electron microscopy, TEM)得到驗證。
Human calcitonin (hCT) is a hormonal peptide composed of 32 amino acids. It plays a crucial role in regulating blood calcium levels in the body by inhibiting the activity of osteoclasts. Therefore, it is often used as a medication to treat bone-related diseases, such as Paget's disease and osteoporosis. Due to its high tendency to aggregate, the efficacy of hCT as a drug is somewhat limited. Our research aims to develop new materials to stabilize this peptide. Previous studies in our laboratory have found that carbohydrate molecules have the potential to inhibit the aggregation of hCT, but they require high concentrations to be effective. In this study, we use the Stöber process to prepare sols and introduce additional glucose, hoping to effectively link the glucose molecules in the solution, thereby reducing the glucose concentration or enhancing the aggregation inhibition effect. In this study, we referred to the preparation conditions in the relevant literature to obtain a stable sample solution, and then optimized the preparation conditions later. Once the stability of the prepared material was improved, we started to adjust the concentration of added glucose. First, we determined the degree of crosslinking of the materials using gel permeation chromatography (GPC) to ensure similar crosslinking levels. We then used Fourier-transform infrared spectroscopy (FTIR) to confirm whether glucose was crosslinked within the materials. Next, we utilized X-ray photoelectron spectroscopy (XPS) to analyze the elemental composition of the materials and estimate the amount of glucose crosslinked within them. In thioflavin T (ThT) kinetic experiments, we observed that the materials inhibited hCT aggregation by reducing the number of hCT fibers when co-incubated. This result was further verified by transmission electron microscopy (TEM).
(1) Chiti, F.; Dobson, C. M. Protein misfolding, amyloid formation, and human disease: a summary of progress over the last decade. Annual Review of Biochemistry 2017, 86, 27-68.
(2) Ross, C. A.; Poirier, M. A. Protein aggregation and neurodegenerative disease. Nature Medicine 2004, 10 Suppl, S10-17.
(3) Abouelasrar Salama, S.; Gouwy, M.; Van Damme, J.; Struyf, S. The turning away of serum amyloid A biological activities and receptor usage. Immunology 2021, 163 (2), 115-127.
(4) Picken, Maria M. The pathology of amyloidosis in classification: a review. Acta Haematologica 2020, 143 (4), 322-334.
(5) Hazenberg, B. P. C. Amyloidosis: a clinical overview. Rheumatic Disease Clinics of North America 2013, 39 (2), 323-345.
(6) Obi, C. A.; Mostertz, W. C.; Griffin, J. M.; Judge, D. P. ATTR epidemiology, genetics, and prognostic factors. Methodist Debakey Cardiovasc J 2022, 18 (2), 17-26.
(7) Sekijima, Y. Transthyretin (ATTR) amyloidosis: clinical spectrum, molecular pathogenesis and disease-modifying treatments. Journal of Neurology, Neurosurgery & Psychiatry 2015, 86 (9), 1036-1043.
(8) Scheltens, P.; De Strooper, B.; Kivipelto, M.; Holstege, H.; Chételat, G.; Teunissen, C. E.; Cummings, J.; van der Flier, W. M. Alzheimer's disease. The Lancet 2021, 397 (10284), 1577-1590.
(9) Ye, H.; Robak, L. A.; Yu, M.; Cykowski, M.; Shulman, J. M. Genetics and pathogenesis of Parkinson's syndrome. Annual Review of Pathology 2023, 18, 95-121.
(10) Pinheiro, L.; Faustino, C. Therapeutic strategies targeting amyloid-β in Alzheimer's disease. Current Alzheimer Research 2019, 16 (5), 418-452.
(11) Cheignon, C.; Tomas, M.; Bonnefont-Rousselot, D.; Faller, P.; Hureau, C.; Collin, F. Oxidative stress and the amyloid beta peptide in Alzheimer's disease. Redox Biology 2018, 14, 450-464.
(12) Mehra, S.; Sahay, S.; Maji, S. K. α-Synuclein misfolding and aggregation: implications in Parkinson's disease pathogenesis. Biochimica et Biophysica Acta 2019, 1867 (10), 890-908.
(13) Chen, R.; Gu, X.; Wang, X. α-Synuclein in Parkinson's disease and advances in detection. Clinica Chimica Acta 2022, 529, 76-86.
(14) Liu, Y.; Yu, P.; Peng, X.; Huang, Q.; Ding, M.; Chen, Y.; Jin, R.; Xie, J.; Zhao, C.; Li, J. Hexapeptide-conjugated calcitonin for targeted therapy of osteoporosis. Journal of Controlled Release 2019, 304, 39-50.
(15) Li, N.; Gong, Y. C.; Chen, J. A meta-analysis of the therapeutic effect of intranasal salmon calcitonin on osteoporosis. European Journal of Medical Research 2021, 26 (1), 140.
(16) Bandeira, L.; Lewiecki, E. M.; Bilezikian, J. P. Pharmacodynamics and pharmacokinetics of oral salmon calcitonin in the treatment of osteoporosis. Expert Opinion on Drug Metabolism & Toxicology 2016, 12 (6), 681-689.
(17) Aliyan, A.; Cook, N. P.; Martí, A. A. Interrogating amyloid aggregates using fluorescent probes. Chemical Reviews 2019, 119 (23), 11819-11856.
(18) Wogulis, M.; Wright, S.; Cunningham, D.; Chilcote, T.; Powell, K.; Rydel, R. E. Nucleation-dependent polymerization is an essential component of amyloid-mediated neuronal cell death. Journal of Neuroscience 2005, 25 (5), 1071-1080.
(19) Lyubchenko, Y. L.; Krasnoslobodtsev, A. V.; Luca, S. Fibrillogenesis of huntingtin and other glutamine containing proteins. Subcellular Biochemistry 2012, 65, 225-251.
(20) Konstantoulea, K.; Louros, N.; Rousseau, F.; Schymkowitz, J. Heterotypic interactions in amyloid function and disease. The FEBS Journal 2022, 289 (8), 2025-2046.
(21) Kristen, A. V. Amyloid cardiomyopathy. Herz 2020, 45 (3), 267-271.
(22) Jin, L.; Gao, W.; Liu, C.; Zhang, N.; Mukherjee, S.; Zhang, R.; Dong, H.; Bhunia, A.; Bednarikova, Z.; Gazova, Z.; et al. Investigating the inhibitory effects of entacapone on amyloid fibril formation of human lysozyme. International Journal of Biological Macromolecules 2020, 161, 1393-1404.
(23) Lippens, G.; Gigant, B. Elucidating tau function and dysfunction in the era of cryo-EM. Journal of Biological Chemistry 2019, 294 (24), 9316-9325.
(24) Jin, L.; Liu, C.; Zhang, N.; Zhang, R.; Yan, M.; Bhunia, A.; Zhang, Q.; Liu, M.; Han, J.; Siebert, H.-C. Attenuation of human lysozyme amyloid fibrillation by ACE Inhibitor captopril: a combined spectroscopy, microscopy, cytotoxicity, and docking study. Biomacromolecules 2021, 22 (5), 1910-1920.
(25) Fitzpatrick, A. W. P.; Falcon, B.; He, S.; Murzin, A. G.; Murshudov, G.; Garringer, H. J.; Crowther, R. A.; Ghetti, B.; Goedert, M.; Scheres, S. H. W. Cryo-EM structures of tau filaments from Alzheimer's disease. Nature 2017, 547 (7662), 185-190.
(26) Copp, D. H.; Cheney, B. Calcitonin-a hormone from the parathyroid which lowers the calcium-level of the blood. Nature 1962, 193, 381-382.
(27) Zaidi, M.; Inzerillo, A. M.; Troen, B.; Moonga, B. S.; Abe, E.; Burckhardt, P. Chapter 82 - molecular and clinical pharmacology of calcitonin. In Principles of Bone Biology Bilezikian, J. P., Raisz, L. G., Rodan, G. A. Eds.; Academic Press, 2002; pp 1423-1440.
(28) calcitonin. https://healthjade.net/calcitonin/, Ed.; 2018.
(29) Boyce, B. F. Advances in the regulation of osteoclasts and osteoclast functions. Journal of Dental Research 2013, 92 (10), 860-867.
(30) Zaidi, M.; Blair, H. C.; Moonga, B. S.; Abe, E.; Huang, C. L. H. Osteoclastogenesis, bone resorption, and osteoclast-based therapeutics. Journal of Bone and Mineral Research 2003, 18 (4), 599-609.
(31) Takito, J.; Inoue, S.; Nakamura, M. The sealing zone in osteoclasts: a self-organized structure on the bone. International Journal of Molecular Sciences 2018, 19 (4).
(32) Bai, R. J.; Li, Y. S.; Zhang, F. J. Osteopontin, a bridge links osteoarthritis and osteoporosis. Frontiers in Endocrinology 2022, 13, 1012508.
(33) Martiniakova, M.; Biro, R.; Kovacova, V.; Babikova, M.; Zemanova, N.; Mondockova, V.; Omelka, R. Current knowledge of bone-derived factor osteocalcin: its role in the management and treatment of diabetes mellitus, osteoporosis, osteopetrosis and inflammatory joint diseases. Journal of Molecular Medicine 2024, 102 (4), 435-452.
(34) Osteoclasts and their role in musculoskeletal disease. https://www.innovationnewsnetwork.com/osteoclasts-and-their-role-in-musculoskeletal-disease/8972/, Ed.; 2021.
(35) Aiyar, N.; Disa, J.; Stadel, J. M.; Lysko, P. G. Calcitonin gene-related peptide receptor independently stimulates 3',5'-cyclic adenosine monophosphate and Ca2+ signaling pathways. Molecular and Cellular Biochemistry 1999, 197 (1-2), 179-185.
(36) Xie, J.; Guo, J.; Kanwal, Z.; Wu, M.; Lv, X.; Ibrahim, N. A.; Li, P.; Buabeid, M. A.; Arafa, E. A.; Sun, Q. Calcitonin and bone physiology: in vitro, in vivo, and clinical investigations. International Journal of Endocrinology 2020, 2020, 3236828.
(37) Kotak, D. J.; Devarajan, P. V. Bone targeted delivery of salmon calcitonin hydroxyapatite nanoparticles for sublingual osteoporosis therapy (SLOT). Nanomedicine 2020, 24, 102153.
(38) Reginster, J. Y. Calcitonin for prevention and treatment of osteoporosis. The America Journal of Medicine 1993, 95 (5a), 44s-47s.
(39) Kanaori, K.; Nosaka, A. Y. Study of human calcitonin fibrillation by proton nuclear magnetic resonance spectroscopy. Biochemistry 1995, 34 (38), 12138-12143.
(40) Itoh-Watanabe, H.; Kamihira-Ishijima, M.; Javkhlantugs, N.; Inoue, R.; Itoh, Y.; Endo, H.; Tuzi, S.; Saitô, H.; Ueda, K.; Naito, A. Role of aromatic residues in amyloid fibril formation of human calcitonin by solid-state 13C NMR and molecular dynamics simulation. Physical Chemistry Chemical Physics 2013, 15 (23), 8890-8901, 10.1039/C3CP44544E.
(41) Yuan, C.; Gao, Z. Aβ interacts with both the iron center and the porphyrin ring of heme: mechanism of heme’s action on Aβ aggregation and disaggregation. Chemical Research in Toxicology 2013, 26 (2), 262-269.
(42) Wu, J.; Zhao, J.; Yang, Z.; Li, H.; Gao, Z. Strong inhibitory effect of heme on hIAPP fibrillation. Chemical Research in Toxicology 2017, 30 (9), 1711-1719.
(43) Ye, H.; Zhou, J.; Li, H.; Gao, Z. Heme prevents highly amyloidogenic human calcitonin (hCT) aggregation: a potential new strategy for the clinical reuse of hCT. Journal of Inorganic Biochemistry 2019, 196, 110686.
(44) Khan, S.; Siraj, S.; Shahid, M.; Haque, M. M.; Islam, A. Osmolytes: wonder molecules to combat protein misfolding against stress conditions. International Journal of Biological Macromolecules 2023, 234, 123662.
(45) Natalello, A.; Liu, J.; Ami, D.; Doglia, S. M.; de Marco, A. The osmolyte betaine promotes protein misfolding and disruption of protein aggregates. Proteins 2009, 75 (2), 509-517.
(46) Burg, M. B.; Ferraris, J. D. Intracellular organic osmolytes: function and regulation*. Journal of Biological Chemistry 2008, 283 (12), 7309-7313.
(47) Venkatraman, A.; Murugan, E.; Lin, S. J.; Peh, G. S. L.; Rajamani, L.; Mehta, J. S. Effect of osmolytes on in-vitro aggregation properties of peptides derived from TGFBIp. Sci Rep 2020, 10 (1), 4011.
(48) Lantz, R.; Busbee, B.; Wojcikiewicz, E. P.; Du, D. Flavonoids with vicinal hydroxyl groups inhibit human calcitonin amyloid formation. Chemistry 2020, 26 (57), 13063-13071.
(49) Zhang, T.-H. Use of sugar osmolytes in inhibiting human calcitonin aggregation. 2023.
(50) Pipattanawarothai, A.; Suksai, C.; Srisook, K.; Trakulsujaritchok, T. Non-cytotoxic hybrid bioscaffolds of chitosan-silica: sol-gel synthesis, characterization and proposed application. Carbohydrate Polymers 2017, 178, 190-199.
(51) Ahaliabadeh, Z.; Kong, X.; Fedorovskaya, E.; Kallio, T. Extensive comparison of doping and coating strategies for Ni-rich positive electrode materials. Journal of Power Sources 2022, 540, 231633.
(52) Tian, Y.; Ran, Z.; Yang, W. Carbon dot-silica composite nanoparticle: an excitation-independent fluorescence material with tunable fluorescence. RSC Advances 2017, 7 (69), 43839-43844, 10.1039/C7RA07990G.
(53) Merrifield, R. B. Solid phase peptide synthesis. I. the synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963, 85 (14), 2149-2154.
(54) Mant, C. T.; Chen, Y.; Yan, Z.; Popa, T. V.; Kovacs, J. M.; Mills, J. B.; Tripet, B. P.; Hodges, R. S. HPLC analysis and purification of peptides. Methods in Molecular Biology 2007, 386, 3-55.
(55) Li, D.; Yi, J.; Han, G.; Qiao, L. MALDI-TOF mass spectrometry in clinical analysis and research. ACS Measurement Science Au 2022, 2 (5), 385-404.
(56) Biancalana, M.; Koide, S. molecular mechanism of thioflavin-T binding to amyloid fibrils. Biochimica et Biophysica Acta 2010, 1804 (7), 1405-1412.
(57) Sahiro, K.; Kawato, Y.; Koike, K.; Sano, T.; Nakai, T.; Sadakane, M. Preyssler-type phosphotungstate is a new family of negative-staining reagents for the TEM observation of viruses. Sci Rep 2022, 12 (1), 7554.
(58) Greasley, S. L.; Page, S. J.; Sirovica, S.; Chen, S.; Martin, R. A.; Riveiro, A.; Hanna, J. V.; Porter, A. E.; Jones, J. R. Controlling particle size in the Stöber process and incorporation of calcium. Journal of Colloid and Interface Science 2016, 469, 213-223.
(59) Al-Saad, K.; Issa, A. A.; Idoudi, S.; Shomar, B.; Al-Ghouti, M. A.; Al-Hashimi, N.; El-Azazy, M. Smart synthesis of trimethyl ethoxysilane (TMS) functionalized core-shell magnetic nanosorbents Fe(3)O(4)@SiO(2): process optimization and application for extraction of pesticides. Molecules 2020, 25 (20).
(60) Li, N.; Lu, W.; Zhu, D. Amino-functionalized silica@resorcinol-formaldehyde nanocomposites for the removal of Cr(VI) from aqueous solutions. Polymers 2023, 15 (20).
(61) Leitgeb, M. Photoelectrochemical porosification of silicon carbide for MEMS. 2020.
(62) Zamani Kouhpanji, M. R.; Stadler, B. A guideline for effectively synthesizing and characterizing magnetic nanoparticles for advancing nanobiotechnology: a review. Sensors 2020, 20, 2554.
(63) Major, G. H.; Fairley, N.; Sherwood, P. M. A.; Linford, M. R.; Terry, J.; Fernandez, V.; Artyushkova, K. Practical guide for curve fitting in x-ray photoelectron spectroscopy. Journal of Vacuum Science & Technology A 2020, 38 (6), 061203.
(64) Priciple of X-ray photoelectron spectroscopy and examples of surface element detection. https://kknews.cc/science/qrp2yyg.html, Ed.; 2022.
(65) Urquhart, R. A. Effects of hot storage on polymer modified binder properties and field performance. 2014.
(66) Chen, Y.-C.; Huang, X.-F.; Hsu, H.-T.; Wu, E.-T.; Peng, C.-H.; Huang, M. H. Photocatalyzed dimethylacrylamide polymerization in an aqueous solution using 4-nitrophenylacetylene-modified Cu2O crystals. Journal of Materials Chemistry A 2024, 12 (24), 14792-14800, 10.1039/D4TA01267D.
(67) Zakirov, A.; Navamathavan, R.; Jang, Y.; Jung, A.; Lee, K.-M.; Choi, C. Comparative study on the structural and electrical properties of low-bftextitk SiOC(-H) films deposited by using plasma enhanced chemical vapor deposition. Journal of The Korean Physical Society - J KOREAN PHYS SOC 2007, 50.