體外仿生肺肝纖維化3D模型研究

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姓名

李奕鋆(Yi-Yun Li) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

論文名稱

體外仿生肺肝纖維化3D模型研究
(Establishment of a 3D dynamic model of in vitro biomimetic lung and liver fibrosis)

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摘要(中)

在人體內組織和細胞會受到不同程度外力刺激，在纖維化過程中機械刺
激很大的影響纖維化程度，且週期性的拉伸可以調控許多的細胞行為，包括
細胞的生長、分化、基因表達和訊息傳遞。另外體內的細胞行為涉及細胞與
周遭微環境之間的動態相互作用，因此本實驗使用甲基丙烯酸酐化魚明膠作
為三維支架模擬體內的微環境進行機械循環拉伸，拉伸條件為應變量 15%、
頻率 0.5 Hz，然後使用商業用矽膠培養膜作為二維培養模型以相同的拉伸條
件比較兩種材料對於纖維化程度的影響，實驗的對象分別是人類肝星狀細胞
株 LX2 以及人類肺成纖維細胞株 HPF，從免疫螢光染色結果發現三維支架
培養的 LX2 和 HPF 在拉伸 24 和 48 小時後肌成纖維標誌物 α-SMA 的表現
量提升，在酵素免疫分析、即時定量聚合酶連鎖反應和西方墨點法也驗證了
三維仿生動態培養更能夠促進細胞纖維化。

摘要(英)

Cells and tissues in the human body are subjected to forces of varying degrees.
During fibrosis, mechanical stimulation greatly affects fibrosis, and cyclic stretch
can regulate many cellular behaviors, including cell growth, differentiation, gene
expression and messaging. In addition, the behavior of cells in vivo involves a
dynamic interaction between cells and the surrounding microenvironment.
Therefore, in this experiment, fish gelatin methacryloyl was used as a threedimensional scaffold to simulate the microenvironment in vivo under mechanical
cyclic stretching. The condition was 15% strain, 0.5 Hz frequency, and then a
polydimethylsiloxane membrane was used as a 2D culture model to compare the
effect of the two materials on the degree of fibrosis under the same stretch
conditions. Human hepatic stellate cell LX2 and human pulmonary fibroblasts HPF
were subjected to cyclic stretching. The results show that immunofluorescent
staining in LX2 and HPF cultured on three-dimensional scaffolds were expressed
more myofibroblast marker α-SMA after stretching for 24 and 48 hours. The results
of enzyme-linked immunosorbent assay, real-time quantitative polymerase chain
reaction and western blot also confirmed that 3D biomimetic dynamic culture is
more conducive to cellular fibrosis.

關鍵字(中)

★ 循環拉伸
★ 3D 模型
★ 纖維化

關鍵字(英)

★ Cyclic Stretch
★ 3D model
★ fibrosis

論文目次

中文摘要 i
Abstract ii
目錄 iii
圖目錄 vii
表目錄 ix
一、緒論 1
1-1纖維化 1
1-1-1肺纖維化 1
1-1-2肝纖維化 2
1-2三維細胞培養模型 3
1-3生物列印 4
1-4三維支架材料 5
1-4-1膠原蛋白(Collagen) 5
1-4-2海藻酸鹽(Alginate) 5
1-4-3透明質酸(Hyaluronic acid) 5
1-4-4甲基丙烯酸酐化明膠(Gelatin methacryloyl) 6
1-5動態培養系統 7
1-6次世代定序(Next Generation Sequencing，NGS) 8
1-7研究動機與目的 8
二、實驗材料與方法 9
2-1實驗方法 9
2-1-1細胞培養 9
2-1-2製備甲基丙烯酸酐化魚明膠(FGelMa) 9
2-1-3甲基丙烯酸酐化魚明膠物化性質分析 10
2-1-3-1流變儀測試 10
2-1-3-2試片強度分析 10
2-1-3-3掃描式電子顯微鏡(SEM) 11
2-1-3-4傅立葉紅外光譜(FTIR) 11
2-1-3-5核磁共振光譜(NMR) 12
2-1-4細胞機械循環拉伸 12
2-1-4-1三維細胞循環拉伸實驗 12
2-1-4-2二維細胞循環拉伸實驗 13
2-1-5細胞毒性分析 14
2-1-6細胞型態及免疫螢光染色 14
2-1-7人類平滑肌動蛋白酵素免疫分析測定 14
2-1-8人類纖連蛋白酵素免疫分析測定 15
2-1-9即時定量聚合酶連鎖反應(RT-qPCR) 15
2-1-10西方墨點法 17
2-1-11 NGS數據分析 17
2-2實驗材料 18
2-2-1細胞培養 18
2-2-2甲基丙烯酸酐化魚明膠材料 18
2-2-3二維機械循環拉伸 19
2-2-4三維機械循環拉伸 19
2-2-5螢光染色實驗 20
2-2-6酵素免疫分析測定套件 20
2-2-7即時定量聚合酶連鎖反應套件 21
2-2-8西方墨點法 21
2-2-9 NGS實驗 23
三、實驗流程 24
四、實驗結果 25
4-1甲基丙烯酸酐化魚明膠物化性質分析 25
4-1-1流變性質 25
4-1-2試片拉伸強度 26
4-1-3 SEM掃描式電子顯微鏡分析 27
4-1-4傅立葉紅外光譜分析 28
4-1-5核磁共振光譜分析 30
4-2細胞毒性分析 31
4-3 HPF細胞機械循環拉伸下的免疫螢光染色 32
4-4 LX2細胞機械循環拉伸下的免疫螢光染色 35
4-5機械循環拉伸後的人類平滑肌動蛋白酵素免疫分析 38
4-6機械循環拉伸後的人類纖連蛋白酵素免疫分析 39
4-7機械循環拉伸後的RT-qPCR結果 40
4-8 HPF細胞機械循環拉伸下的western blot結果 41
4-9 LX2細胞機械循環拉伸下的western blot結果 42
4-10經由機械循環拉伸的HPF和LX2細胞主成分分析圖 43
4-11經由機械循環拉伸的HPF和LX2細胞相關性矩陣 45
4-12經由機械循環拉伸的細胞和靜態培養的基因表達譜 47
4-13經由機械循環拉伸後表現量有交集的差異表達基因 50
4-14差異表現基因的訊號通路分析 51
五、討論與結論 54
5-1特定條件的機械循環拉伸可以使纖維化細胞的α-SMA蛋白表現量上升 54
5-2以酵素免疫分析、qPCR以及西方墨點法佐證機械循環拉伸對纖維化細胞的影響 54
5-3探討機械循環拉伸下HPF和LX2細胞差異表達基因顯著相關的訊號通路 55
5-4結論 60
參考文獻 61
附錄 66

參考文獻

1. Quan, T.E., S.E. Cowper, and R. Bucala, The role of circulating fibrocytes in fibrosis. Curr Rheumatol Rep, 2006. 8(2): p. 145-50.
2. Willis, B.C., R.M. duBois, and Z. Borok, Epithelial origin of myofibroblasts during fibrosis in the lung. Proc Am Thorac Soc, 2006. 3(4): p. 377-82.
3. Kalluri, R. and E.G. Neilson, Epithelial-mesenchymal transition and its implications for fibrosis. J Clin Invest, 2003. 112(12): p. 1776-84.
4. Wynn, T.A., Cellular and molecular mechanisms of fibrosis. J Pathol, 2008. 214(2): p. 199-210.
5. Henderson, N.C., F. Rieder, and T.A. Wynn, Fibrosis: from mechanisms to medicines. Nature, 2020. 587(7835): p. 555-566.
6. Salton, F., M.C. Volpe, and M. Confalonieri, Epithelial⁻Mesenchymal Transition in the Pathogenesis of Idiopathic Pulmonary Fibrosis. Medicina (Kaunas), 2019. 55(4).
7. Lu, Q. and A.H.K. El-Hashash, Cell-based therapy for idiopathic pulmonary fibrosis. Stem Cell Investig, 2019. 6: p. 22.
8. Zoubek, M.E., C. Trautwein, and P. Strnad, Reversal of liver fibrosis: From fiction to reality. Best Pract Res Clin Gastroenterol, 2017. 31(2): p. 129-141.
9. Simon, T.G., et al., Non-alcoholic fatty liver disease in children and young adults is associated with increased long-term mortality. J Hepatol, 2021. 75(5): p. 1034-1041.
10. Abutaleb, A., et al., Higher Levels of Fibrosis in a Cohort of Veterans with Chronic Viral Hepatitis are Associated with Extrahepatic Cancers. J Clin Exp Hepatol, 2021. 11(2): p. 195-200.
11. Zhang, M., et al., Hepatic stellate cell senescence in liver fibrosis: Characteristics, mechanisms and perspectives. Mech Ageing Dev, 2021. 199: p. 111572.
12. Iredale, J.P., Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest, 2007. 117(3): p. 539-48.
13. Elpek, G., Cellular and molecular mechanisms in the pathogenesis of liver fibrosis: An update. World J Gastroenterol, 2014. 20(23): p. 7260-76.
14. Roehlen, N., E. Crouchet, and T.F. Baumert, Liver Fibrosis: Mechanistic Concepts and Therapeutic Perspectives. Cells, 2020. 9(4).
15. Birgersdotter, A., R. Sandberg, and I. Ernberg, Gene expression perturbation in vitro--a growing case for three-dimensional (3D) culture systems. Semin Cancer Biol, 2005. 15(5): p. 405-12.
16. Edmondson, R., et al., Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol, 2014. 12(4): p. 207-18.
17. Baruffaldi, D., et al., 3D Cell Culture: Recent Development in Materials with Tunable Stiffness. ACS Appl Bio Mater, 2021. 4(3): p. 2233-2250.
18. Trédan, O., et al., Drug resistance and the solid tumor microenvironment. J Natl Cancer Inst, 2007. 99(19): p. 1441-54.
19. Ho, W.J., et al., Incorporation of multicellular spheroids into 3-D polymeric scaffolds provides an improved tumor model for screening anticancer drugs. Cancer Sci, 2010. 101(12): p. 2637-43.
20. Langhans, S.A., Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning. Front Pharmacol, 2018. 9: p. 6.
21. Fang, Y. and R.M. Eglen, Three-Dimensional Cell Cultures in Drug Discovery and Development. SLAS Discov, 2017. 22(5): p. 456-472.
22. Groll, J., et al., Biofabrication: reappraising the definition of an evolving field. Biofabrication, 2016. 8(1): p. 013001.
23. Ozbolat, I.T. and M. Hospodiuk, Current advances and future perspectives in extrusion-based bioprinting. Biomaterials, 2016. 76: p. 321-43.
24. Gudapati, H., M. Dey, and I. Ozbolat, A comprehensive review on droplet-based bioprinting: Past, present and future. Biomaterials, 2016. 102: p. 20-42.
25. Klak, M., et al., Novel Strategies in Artificial Organ Development: What Is the Future of Medicine? Micromachines (Basel), 2020. 11(7).
26. Mandrycky, C., et al., 3D bioprinting for engineering complex tissues. Biotechnol Adv, 2016. 34(4): p. 422-434.
27. Kolesky, D.B., et al., 3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs. Adv Mater, 2014. 26(19): p. 3124-30.
28. Chattopadhyay, S. and R.T. Raines, Review collagen-based biomaterials for wound healing. Biopolymers, 2014. 101(8): p. 821-33.
29. Osidak, E.O., et al., Viscoll collagen solution as a novel bioink for direct 3D bioprinting. J Mater Sci Mater Med, 2019. 30(3): p. 31.
30. Rowley, J.A., G. Madlambayan, and D.J. Mooney, Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials, 1999. 20(1): p. 45-53.
31. Lee, K.Y. and D.J. Mooney, Alginate: properties and biomedical applications. Prog Polym Sci, 2012. 37(1): p. 106-126.
32. Gu, Z., et al., Development of 3D bioprinting: From printing methods to biomedical applications. Asian J Pharm Sci, 2020. 15(5): p. 529-557.
33. Jia, J., et al., Engineering alginate as bioink for bioprinting. Acta Biomater, 2014. 10(10): p. 4323-31.
34. Burdick, J.A., et al., Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks. Biomacromolecules, 2005. 6(1): p. 386-91.
35. Sherman, L., et al., Hyaluronate receptors: key players in growth, differentiation, migration and tumor progression. Curr Opin Cell Biol, 1994. 6(5): p. 726-33.
36. Kobayashi, Y., A. Okamoto, and K. Nishinari, Viscoelasticity of hyaluronic acid with different molecular weights. Biorheology, 1994. 31(3): p. 235-44.
37. Yoon, H.J., et al., Cold Water Fish Gelatin Methacryloyl Hydrogel for Tissue Engineering Application. PLoS One, 2016. 11(10): p. e0163902.
38. Sun, M., et al., Synthesis and Properties of Gelatin Methacryloyl (GelMA) Hydrogels and Their Recent Applications in Load-Bearing Tissue. Polymers (Basel), 2018. 10(11).
39. Ma, C., et al., Mammalian and Fish Gelatin Methacryloyl-Alginate Interpenetrating Polymer Network Hydrogels for Tissue Engineering. ACS Omega, 2021. 6(27): p. 17433-17441.
40. Zhang, X., et al., Marine Biomaterial-Based Bioinks for Generating 3D Printed Tissue Constructs. Mar Drugs, 2018. 16(12).
41. Nichol, J.W., et al., Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials, 2010. 31(21): p. 5536-44.
42. Leu Alexa, R., et al., 3D-Printed Gelatin Methacryloyl-Based Scaffolds with Potential Application in Tissue Engineering. Polymers (Basel), 2021. 13(5).
43. Young, A.T., O.C. White, and M.A. Daniele, Rheological Properties of Coordinated Physical Gelation and Chemical Crosslinking in Gelatin Methacryloyl (GelMA) Hydrogels. Macromol Biosci, 2020. 20(12): p. e2000183.
44. Geiger, R.C., et al., Cyclic stretch-induced reorganization of the cytoskeleton and its role in enhanced gene transfer. Gene Ther, 2006. 13(8): p. 725-31.
45. Chhetri, A., J.V. Rispoli, and S.A. Lelièvre, 3D Cell Culture for the Study of Microenvironment-Mediated Mechanostimuli to the Cell Nucleus: An Important Step for Cancer Research. Front Mol Biosci, 2021. 8: p. 628386.
46. Balestrini, J.L. and K.L. Billiar, Equibiaxial cyclic stretch stimulates fibroblasts to rapidly remodel fibrin. J Biomech, 2006. 39(16): p. 2983-90.
47. Asmani, M., et al., Cyclic Stretching of Fibrotic Microtissue Array for Evaluation of Anti-Fibrosis Drugs. Cell Mol Bioeng, 2019. 12(5): p. 529-540.
48. Cui, Y., et al., Cyclic stretching of soft substrates induces spreading and growth. Nat Commun, 2015. 6: p. 6333.
49. Faust, U., et al., Cyclic stress at mHz frequencies aligns fibroblasts in direction of zero strain. PLoS One, 2011. 6(12): p. e28963.
50. Inoh, H., et al., Uni-axial cyclic stretch induces the activation of transcription factor nuclear factor kappaB in human fibroblast cells. Faseb j, 2002. 16(3): p. 405-7.
51. Sanger, F., S. Nicklen, and A.R. Coulson, DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A, 1977. 74(12): p. 5463-7.
52. Yohe, S. and B. Thyagarajan, Review of Clinical Next-Generation Sequencing. Arch Pathol Lab Med, 2017. 141(11): p. 1544-1557.
53. Mardis, E.R., Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet, 2008. 9: p. 387-402.
54. Nijman, I.J., et al., Targeted next-generation sequencing: a novel diagnostic tool for primary immunodeficiencies. J Allergy Clin Immunol, 2014. 133(2): p. 529-34.
55. Meyerson, M., S. Gabriel, and G. Getz, Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet, 2010. 11(10): p. 685-96.
56. Warren, K.M., et al., 2D and 3D Mechanobiology in Human and Nonhuman Systems. ACS Appl Mater Interfaces, 2016. 8(34): p. 21869-82.
57. Tomasek, J.J., et al., Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol, 2002. 3(5): p. 349-63.
58. Kolahian, S., et al., Immune Mechanisms in Pulmonary Fibrosis. Am J Respir Cell Mol Biol, 2016. 55(3): p. 309-22.
59. Xu, R., Z. Zhang, and F.S. Wang, Liver fibrosis: mechanisms of immune-mediated liver injury. Cell Mol Immunol, 2012. 9(4): p. 296-301.
60. Wynn, T.A., Integrating mechanisms of pulmonary fibrosis. J Exp Med, 2011. 208(7): p. 1339-50.
61. Barron, L. and T.A. Wynn, Fibrosis is regulated by Th2 and Th17 responses and by dynamic interactions between fibroblasts and macrophages. Am J Physiol Gastrointest Liver Physiol, 2011. 300(5): p. G723-8.
62. Moore, B.B., et al., CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury. Am J Pathol, 2005. 166(3): p. 675-84.
63. Lin, C.H., et al., CXCL12 induces connective tissue growth factor expression in human lung fibroblasts through the Rac1/ERK, JNK, and AP-1 pathways. PLoS One, 2014. 9(8): p. e104746.
64. Wu, L., et al., Quercetin prevents hepatic fibrosis by inhibiting hepatic stellate cell activation and reducing autophagy via the TGF-β1/Smads and PI3K/Akt pathways. Sci Rep, 2017. 7(1): p. 9289.
65. Wynn, T.A. and T.R. Ramalingam, Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med, 2012. 18(7): p. 1028-40.
66. Wynn, T.A., Fibrotic disease and the T(H)1/T(H)2 paradigm. Nat Rev Immunol, 2004. 4(8): p. 583-94.
67. Lamouille, S., J. Xu, and R. Derynck, Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol, 2014. 15(3): p. 178-96.
68. Gonzalez, D.M. and D. Medici, Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal, 2014. 7(344): p. re8.
69. Roeb, E., Matrix metalloproteinases and liver fibrosis (translational aspects). Matrix Biol, 2018. 68-69: p. 463-473.

指導教授

許藝瓊(Yi-Chiung Hsu)

審核日期

2022-8-20

推文