博碩士論文 111324041 詳細資訊



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：70 、訪客IP：3.145.44.216
姓名 蔡弘源(HUNG-YUAN TSAI)  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 利用組織工程製作仿生腫瘤 並以MRI進行影像分析
(Preparation of A Bionic Tumor using Tissue Engineering and Its Image Analysis by MRI)
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) 本研究針對不同腫瘤細胞接種方法，在三維明膠/羥基磷灰石/戊二醛（GHG）支架中所形成腫瘤仿體。利用被動接種的蓋壓法，以及主動接種的培養液注射法和明膠注射法將細胞植入支架中，並通過磁共振成像（MRI）觀察腫瘤組織的生長情況。結果顯示，蓋壓法會導致細胞主要集中於細胞液接觸面；培養液注射法中，細胞能分布於支架的注射上層與注射層，而注射下層的細胞液可能因重力作用而流失。明膠注射法中，觀察到支架的注射上層、注射層與注射下層的孔洞內皆有細胞被明膠包覆，並推測黏性的明膠能將細胞維持在孔洞內進行懸浮生長。在14天的培養後，三種接種方法在表層均能培養出緻密的組織團塊，其中明膠注射法不僅可以增加在組織團塊數，也能促進在支架縱向的分布。在MRI觀察中，影像解析度的限制為83 μm，因此無法成像較小的腫瘤組織或孔洞，但MRI能對腫瘤組織在支架的分布進行大尺度的觀察。此外，MRI影像能進行三維重建，但支架結構與腫瘤組織的訊號存在重疊，導致兩者無法清晰區分。未來研究可考慮對MRI影像進行優化處理，並對仿體進行連續觀察。
摘要(英) This study investigates the impact of different cell seeding methods on the formation of bionic tumors in a three-dimensional GHG（gelatin/hydroxyapatite/glutaraldehyde） scaffold. Three seeding methods were evaluated including passive seeding by capping and active seeding via culture medium injection and gelatin injection. Capping method allowed cell suspension passively absorbed to the scaffold, leading to cell primarily concentrated on the cell suspension-contacting surface. In the culture medium injection method, cells were distributed in the injection layer and its upper. Cell was unfindable in the region below the injection layer due to possible lost through gravity. In contrast, with the gelatin injection method, cells were observed encapsulated by gelatin within the pores not only in the injection layer but also its upper and lower regions, suggesting that gelatin′s supportive properties enabled cell suspended growth within the pores. After cultivation for 2 weeks, all seeding methods resulted in the formation of dense tissue, with a significant increase in vertical growth area observed in the gelatin injection method, indicating that the suspension culture in gelatin could promote tissue growth in the vertical dimension. These bionic tumor were monitored using magnetic resonance imaging（MRI）analysis.Due to the resolution limitation of 83 μm of MRI, it was difficult to image smaller tumor tissues or pores within the scaffold. However, MRI could conduct large-scale observations of the distribution of tumor tissue in the scaffold. The 3D reconstruction results of MRI showed that the signals of the scaffold structure and the tumor tissue overlaped, preventing clear distinction between the two. Future research could focus on optimizing MRI imaging and continuously monitoring tissue growth in the scaffold.
關鍵字(中) ★ 組織工程
★ 核磁共振影像		 關鍵字(英)
論文目次 摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 V
表目錄 VI
第一章、緒論 1
1.1 現今概況 1
1.2 研究動機與挑戰 2
1.3 研究方法與目的 3
第二章、理論基礎 4
2.1癌症骨轉移 4
2.1.1 臨床上骨轉移的治療 5
2.1.2癌症骨轉移機制 6
2.1.3表皮生長因子對於骨轉移的影響 7
2.2動物模型 9
2.3 腫瘤組織體外培養 11
2.3.1 二維培養 11
2.3.2 腫瘤的細胞外基質 12
2.3.3 體外研究腫瘤大小 13
2.4 組織工程 15
2.4.1仿骨支架 16
2.4.2 體外支架材料 17
2.4.3 體外支架的細胞接種方式 19
2.4.4細胞維持在三維孔洞 21
2.4.5 體外三維仿體的培養 23
2.5腫瘤觀察方式 24
2.5.1侵入式觀察 26
2.5.2非侵入式觀察 28
2.5.2.1 MRI及MRI成像原理 30
第三章、材料與方法 32
3.1實驗藥品 32
3.2實驗儀器 33
3.3實驗流程圖 34
3.4胰臟癌細胞 35
3.4.1 胰臟癌細胞選擇與來源 35
3.4.2胰臟癌細胞繼代 36
3.5體外胰臟癌仿體 38
3.5.1仿骨支架製備 38
3.5.2製備骨轉移微環境 43
3.6胰臟癌與仿骨支架培養 48
3.6.1支架接種胰臟癌細胞 48
3.6.2計算三維仿體上的細胞數量 50
3.6.3三維動態培養系統 51
3.6.4仿體動態培養系統 53
3.7胰臟癌骨轉移仿體分析 54
3.7.1細胞假體 54
3.7.2 MRI 55
3.7.3 MRI影像分析/處理 56
3.7.4組織切片分析 61
第四章、 結果 63
4.1三維仿骨支架之特性 63
4.1.1三維仿骨支架結構與孔徑 63
4.2細胞接種 66
4.2.1蓋壓法 67
4.2.1.1蓋壓法0天的細胞垂直分布 67
4.2.1.2蓋壓法0天的細胞水平面分布 70
4.2.2 培養液注射法 71
4.2.2.1 培養液注射法0天的細胞矢狀分布 71
4.2.2.2 培養液注射法0天的細胞水平面分布 74
4.2.3 明膠注射法 75
4.2.3.1 明膠注射法0天的細胞垂直分布 75
4.2.3.2 明膠注射法0天的細胞水平面分布 78
4.3 腫瘤細胞於三維支架培養14天 80
4.3.1 蓋壓法 80
4.3.1.1蓋壓法培養後細胞的矢狀生長 80
4.3.1.2 蓋壓法培養後細胞的水平面生長 83
4.3.2 培養液注射法 85
4.3.2.1 培養液注射法培養後細胞的矢狀生長 85
4.3.2.2 培養液注射法培養後細胞的水平面生長 88
4.3.3 明膠注射法 89
4.3.3.1 明膠注射法培養後細胞的矢狀生長 89
4.3.3.2 明膠注射法培養後細胞的水平生長 93
4.3.4不同注射方法對於組織生長的比較 95
4.4 MRI的觀察 96
4.4.1 MRI平面影像上觀察組織 97
4.4.2 MRI二維影像中組織可視化提升 99
4.4.3 組織切片與MRI比較 101
4.4.4 MRI三維影像重建 105
第五章、 結論 110
第六章、 展望 112
第七章、 參考文獻 114
第八章、附錄 120
參與研討會與發表 120
參考文獻 1. Bosetti, C., et al., Pancreatic cancer: Overview of descriptive epidemiology. Molecular Carcinogenesis, 2012. 51(1). 3-13.
2. Leowattana, W., P. Leowattana, and T. Leowattana, Systemic treatment for advanced pancreatic cancer. World J Gastrointest Oncol, 2023. 15(10). 1691-1705.
3. Cassalia, F., et al., The Importance of Reading the Skin: Cutaneous Metastases of Pancreatic Cancer, a Systematic Review. Journal of Clinical Medicine, 2024. 13(1). 104.
4. Park, B.K., et al., Suspicious findings observed retrospectively on CT imaging performed before the diagnosis of pancreatic cancer. J Gastrointest Oncol, 2023. 14(2). 1008-1018.
5. Fitzgerald, K.A., et al., Life in 3D is never flat: 3D models to optimise drug delivery. Journal of Controlled Release, 2015. 215. 39-54.
6. Pahouja, G., et al., Stabilization of bone marrow infiltration by metastatic breast cancer with continuous doxorubicin. Cancer Treatment Communications, 2015. 3. 28-32.
7. Dittus, C., et al., Bone marrow infiltration as the initial presentation of gastric signet ring cell adenocarcinoma. Journal of Gastrointestinal Oncology, 2014. 5(6). E113-E116.
8. Yang, H., et al., Clinical features and treatment of bone marrow metastasis. Oncol Lett, 2023. 26(2). 332.
9. Rong, T., et al., A Rare Manifestation of a Presumed Non-Osteophilic Brain Neoplasm: Extensive Axial Skeletal Metastases From Glioblastoma With Primitive Neuronal Components. Frontiers in Oncology, 2021. 11. 760697.
10. Massagué, J. and A.C. Obenauf, Metastatic colonization by circulating tumour cells. Nature, 2016. 529(7586). 298-306.
11. Saxena, M. and G. Christofori, Rebuilding cancer metastasis in the mouse. Molecular Oncology, 2013. 7(2). 283-296.
12. Carpenter, G. and S. Cohen, Epidermal Growth Factor. Annual Review of Biochemistry, 1979. 48(Volume 48, 1979). 193-216.
13. Bowman, B., FADD and its Phosphorylation Mediate Mitogenic Signaling in Mutant Kras Tumors. 2015. 395–401.
14. Zhu, G., et al., Bone physiological microenvironment and healing mechanism: Basis for future bone-tissue engineering scaffolds. Bioactive Materials, 2021. 6(11). 4110-4140.
15. Huang, C.-J., et al. A Tumor Accelerator Based on Multicomponent Bone Scaffolds and Cancer Cell Homing. Polymers, 2022. 14. 14163340.
16. Liu, T., et al., Alendronate-Modified Polymeric Micelles for the Treatment of Breast Cancer Bone Metastasis. Molecular Pharmaceutics, 2019. 16(7). 2872-2883.
17. Mak, I.W., N. Evaniew, and M. Ghert, Lost in translation: animal models and clinical trials in cancer treatment. Am J Transl Res, 2014. 6(2). 114-8.
18. Brancato, V., et al., Could 3D models of cancer enhance drug screening? Biomaterials, 2020. 232. 119744.
19. DesRochers, T.M., E. Palma, and D.L. Kaplan, Tissue-engineered kidney disease models. Advanced Drug Delivery Reviews, 2014. 69-70. 67-80.
20. Hinderer, S., S.L. Layland, and K. Schenke-Layland, ECM and ECM-like materials — Biomaterials for applications in regenerative medicine and cancer therapy. Advanced Drug Delivery Reviews, 2016. 97. 260-269.
21. Frantz, C., K.M. Stewart, and V.M. Weaver, The extracellular matrix at a glance. Journal of Cell Science, 2010. 123(24). 4195-4200.
22. De Jaeghere, E., et al., Heterocellular 3D scaffolds as biomimetic to recapitulate the tumor microenvironment of peritoneal metastases in vitro and in vivo. Biomaterials, 2018. 158. 95-105.
23. Florczyk, S.J., et al., 3D Porous Chitosan–Alginate Scaffolds: A New Matrix for Studying Prostate Cancer Cell–Lymphocyte Interactions In Vitro. Advanced Healthcare Materials, 2012. 1(5). 590-599.
24. Weaver, V.M., et al., Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies. J Cell Biol, 1997. 137(1). 231-45.
25. Erler, J.T., et al., Lysyl oxidase is essential for hypoxia-induced metastasis. Nature, 2006. 440(7088). 1222-1226.
26. Maitra Roy, S., et al., 3D multicellular tumor spheroids used for in vitro preclinical therapeutic screening. Journal of Drug Delivery Science and Technology, 2023. 86. 104636.
27. Kingsley, D.M., et al., Laser-based 3D bioprinting for spatial and size control of tumor spheroids and embryoid bodies. Acta Biomaterialia, 2019. 95. 357-370.
28. Saxena, A., Tissue engineering: Present concepts and strategies. Journal of Indian Association of Pediatric Surgeons, 2005. 10. 14-19.
29. Wang, H., et al., Fabrication and properties of hydroxyapatite/chitosan composite scaffolds loaded with periostin for bone regeneration. Heliyon, 2024. 10(5). e25832.
30. Białkowska, K., et al. Spheroids as a Type of Three-Dimensional Cell Cultures—Examples of Methods of Preparation and the Most Important Application. International Journal of Molecular Sciences, 2020. 21. 21176225.
31. Mehta, G., et al., Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. Journal of Controlled Release, 2012. 164(2). 192-204.
32. Lamb, R., et al., Co-ordination of cell cycle, migration and stem cell-like activity in breast cancer. Oncotarget, 2014. 5(17). 7833–7842
33. Xu, X., M.C. Farach-Carson, and X. Jia, Three-dimensional in vitro tumor models for cancer research and drug evaluation. Biotechnology Advances, 2014. 32(7) 1256-1268.
34. Antunes, J., et al., In-air production of 3D co-culture tumor spheroid hydrogels for expedited drug screening. Acta Biomaterialia, 2019. 94. 392-409.
35. Afewerki, S., et al., Gelatin-polysaccharide composite scaffolds for 3D cell culture and tissue engineering: Towards natural therapeutics. Bioengineering & Translational Medicine, 2019. 4(1). 96-115.
36. Alipal, J., et al., A review of gelatin: Properties, sources, process, applications, and commercialisation. Materials Today: Proceedings, 2021. 42. 240-250.
37. Michelini, L., et al., Characterization of gelatin hydrogels derived from different animal sources. Materials Letters, 2020. 272. 127865.
38. Zhao, X., et al., Photocrosslinkable gelatin hydrogel for epidermal tissue engineering. Advanced healthcare materials, 2016. 5(1). 108-118.
39. Rijal, G. and W. Li, A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening. Science advances, 2017. 3(9). e1700764.
40. Zhanbassynova, A., et al. Impact of Hydroxyapatite on Gelatin/Oxidized Alginate 3D-Printed Cryogel Scaffolds. Gels, 2024. 10. 406.
41. Cheng, C.-H., et al., Immobilization of bone morphogenetic protein-2 to gelatin/avidin-modified hydroxyapatite composite scaffolds for bone regeneration. Journal of Biomaterials Applications, 2019. 33(9). 1147-1156.
42. Sanz-Herrera, J.A., J.M. García-Aznar, and M. Doblaré, On scaffold designing for bone regeneration: A computational multiscale approach. Acta Biomaterialia, 2009. 5(1). 219-229.
43. Shi, F., et al., Improved cell seeding efficiency and cell distribution in porous hydroxyapatite scaffolds by semi-dynamic method. Cell and Tissue Banking, 2022. 23(2). 313-324.
44. Sobral, J.M., et al., Three-dimensional plotted scaffolds with controlled pore size gradients: Effect of scaffold geometry on mechanical performance and cell seeding efficiency. Acta Biomaterialia, 2011. 7(3). 1009-1018.
45. Zhang, Z.-Z., et al., Potential of Centrifugal Seeding Method in Improving Cells Distribution and Proliferation on Demineralized Cancellous Bone Scaffolds for Tissue-Engineered Meniscus. 2022. 15294-15302.
46. Cámara-Torres, M., et al., Improving cell distribution on 3D additive manufactured scaffolds through engineered seeding media density and viscosity. Acta Biomaterialia, 2020. 101. 183-195.
47. Alvarez-Barreto, J.F., et al., Flow Perfusion Improves Seeding of Tissue Engineering Scaffolds with Different Architectures. Annals of Biomedical Engineering, 2007. 35(3). 429-442.
48. Zhang, Z.-Z., et al., Potential of Centrifugal Seeding Method in Improving Cells Distribution and Proliferation on Demineralized Cancellous Bone Scaffolds for Tissue-Engineered Meniscus. ACS Applied Materials & Interfaces, 2015. 7(28). 15294-15302.
49. Olivares, A.L. and D. Lacroix, Simulation of Cell Seeding Within a Three-Dimensional Porous Scaffold: A Fluid-Particle Analysis. Tissue Engineering Part C: Methods, 2012. 18(8). 624-631.
50. Cheng, G., et al., Cell Population Dynamics Modulate the Rates of Tissue Growth Processes. Biophysical Journal, 2006. 90(3). 713-724.
51. Bueno, E.M., G. Laevsky, and G.A. Barabino, Enhancing cell seeding of scaffolds in tissue engineering through manipulation of hydrodynamic parameters. Journal of Biotechnology, 2007. 129(3). 516-531.
52. Karami, D., N. Richbourg, and V. Sikavitsas, Dynamic in vitro models for tumor tissue engineering. Cancer Letters, 2019. 449. 178-185.
53. Cheng, G., P. Markenscoff, and K. Zygourakis, A 3D Hybrid Model for Tissue Growth: The Interplay between Cell Population and Mass Transport Dynamics. Biophysical Journal, 2009. 97(2). 401-414.
54. McCorry, M.C., et al., Sensor technologies for quality control in engineered tissue manufacturing. Biofabrication, 2023. 15(1). 012001.
55. Appel, A.A., et al., Imaging challenges in biomaterials and tissue engineering. Biomaterials, 2013. 34(28). 6615-6630.
56. Gordillo, N., E. Montseny, and P. Sobrevilla, State of the art survey on MRI brain tumor segmentation. Magnetic Resonance Imaging, 2013. 31(8). 1426-1438.
57. Washburn, N.R., et al., Bone formation in polymeric scaffolds evaluated by proton magnetic resonance microscopy and X-ray microtomography. Journal of Biomedical Materials Research Part A, 2004. 69A(4). 738-747.
58. Leferink, A.M., et al., An Open Source Image Processing Method to Quantitatively Assess Tissue Growth after Non-Invasive Magnetic Resonance Imaging in Human Bone Marrow Stromal Cell Seeded 3D Polymeric Scaffolds. PLOS ONE, 2014. 9(12). e115000.
59. Takagi, K., et al., Analysis of microvessels in pancreatic cancer: by light microscopy, confocal laser scan microscopy, and electron microscopy. Journal of Hepato-Biliary-Pancreatic Surgery, 2008. 15(4). 384-390.
60. Caicedo, J.C., et al., Evaluation of Deep Learning Strategies for Nucleus Segmentation in Fluorescence Images. Cytometry Part A, 2019. 95(9). 952-965.
61. Oprea-Lager, D.E., et al., Bone Metastases Are Measurable: The Role of Whole-Body MRI and Positron Emission Tomography. Frontiers in Oncology, 2021. 11. 772530
62. Van der Wall, E., et al., Diagnostic significance of gadolinium-DTPA (diethylenetriamine penta-acetic acid) enhanced magnetic resonance imaging in thrombolytic treatment for acute myocardial infarction: its potential in assessing reperfusion. Heart, 1990. 63(1). 12-17.
63. Onoue, K., et al., Temporal subtraction of computed tomography images improves detectability of bone metastases by radiology residents. European radiology, 2019. 1-4.
64. Morsing, A., et al., Hybrid PET/MRI in major cancers: a scoping review. European journal of nuclear medicine and molecular imaging, 2019. 1-14.
65. Matani, H., et al., Utilization of functional MRI in the diagnosis and management of cervical cancer. Frontiers in Oncology, 2022. 12. 1030967.
66. Nagesh, C.P., et al., Magnetic resonance imaging of the orbit, Part 1: Basic principles and radiological approach. Indian Journal of Ophthalmology, 2021. 69(10). 2574– 2584
67. Belaroussi, B., et al., Intensity non-uniformity correction in MRI: Existing methods and their validation. Medical Image Analysis, 2006. 10(2). 234-246.
68. Cha, D.I., et al., Pancreatic ductal adenocarcinoma: Prevalence and diagnostic value of dark choledochal ring sign on T2-weighted MRI. Clinical Radiology, 2014. 69(4). 416-423.
69. Lieber, M., et al., Establishment of a continuous tumor-cell line (PANC-1) from a human carcinoma of the exocrine pancreas. International Journal of Cancer, 1975. 15(5). 741-747.
70. Young, S., et al., Gelatin as a delivery vehicle for the controlled release of bioactive molecules. Journal of Controlled Release, 2005. 109(1). 256-274.
71. Gupta, S.K., et al., Modification of decellularized goat-lung scaffold with chitosan/nanohydroxyapatite composite for bone tissue engineering applications. BioMed research international, 2013. 2013. 651945.
72. Youn, B.S., et al., Large-Scale Expansion of Mammary Epithelial Stem Cell Aggregates in Suspension Bioreactors. Biotechnology Progress, 2005. 21(3). 984-993.
73. Chen, K.-Y., et al., Autologous bone marrow stromal cells loaded onto porous gelatin scaffolds containing Drynaria fortunei extract for bone repair. Journal of Biomedical Materials Research Part A, 2013. 101A(4). 954-962.
74. Kubíková, T., et al., Comparison of ground sections, paraffin sections and micro-CT imaging of bone from the epiphysis of the porcine femur for morphometric evaluation. Annals of Anatomy - Anatomischer Anzeiger, 2018. 220. 85-96.
75. Chang, C.C., et al., Direct-write bioprinting three-dimensional biohybrid systems for future regenerative therapies. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2011. 98B(1). 160-170.
76. Reznikov, N., et al., The 3D structure of the collagen fibril network in human trabecular bone: Relation to trabecular organization. Bone, 2015. 71. 189-195.
77. Abarrategi, A., et al., Label-free magnetic resonance imaging to locate live cells in three-dimensional porous scaffolds. Journal of The Royal Society Interface, 2012. 9(74). 2321-2331.
78. Sinibaldi, R., et al., Multimodal-3D imaging based on μMRI and μCT techniques bridges the gap with histology in visualization of the bone regeneration process. Journal of Tissue Engineering and Regenerative Medicine, 2018. 12(3). 750-761.
79. Crowe, J.J., et al., A magnetic resonance-compatible perfusion bioreactor system for three-dimensional human mesenchymal stem cell construct development. Chemical Engineering Science, 2011. 66(18). 4138-4147.
指導教授 胡威文 董國忠(Wei-Wen Hu Guo-Chung Dong) 審核日期 2024-9-20
推文 facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu   
網路書籤 Google bookmarks   del.icio.us   hemidemi   myshare   

若有論文相關問題，請聯絡國立中央大學圖書館推廣服務組 TEL:(03)422-7151轉57407，或E-mail聯絡  - 隱私權政策聲明