全世界生產超過 3.5 億噸塑膠； 最終並釋放到環境中的塑膠會被降解為微米塑膠/奈米塑膠。與塑膠塊材相比，奈米塑膠對健康的影響仍未被充分探索。在這項研究中，我們使用微流道裝置建立了一個多層模型來重建上皮屏障，以研究奈米塑膠對上皮屏障的影響。為此，研究中所開發與使用的工程技術方法具有多項優點，(1)三維細胞培養: 相較於傳統二維細胞培養，三維細胞培養發法提供更接近生理條件的微環境、氧氣和營養梯度、以及細胞間的相互作用與細胞外培養基 (ECM)。(2)開發一體外皮膚等效模型:能應用於高通量藥物篩檢，以替代昂貴、耗時，並且有衍生道德問題的動物試驗。(3) 新型微流細胞培養晶片:利用生物相容性的聚碳酸酯(polycarbonate)不須額外設備和複雜的固定包埋程序，即可進行多層細胞培養，同時能即時觀察量測多層細胞樣本的側面切片影像。我們利用代表表皮和真皮的人角質形成細胞和成纖維細胞逐層共培養，建立模擬臨床切片空間分布之多層細胞模型(上表皮層約0.1-0.2mm，和上真皮層約0.3-0.6mm。）。將奈米塑膠施予於細胞層上，我們發現細胞間的緊密連接發生了顯著型態改變； 奈米塑膠其表面特性影響其可滲透至多細胞結構之深度。我們觀察到細胞內活性氧(ROS)在細胞內部過量累積，表示奈米塑膠可能導致粒線體功能障礙，進而影響細胞內重要的生理功能。透過我們開發的體外組織模型，可以進一步了解奈米塑膠對人類表皮組織的影響。而我們的數據也表明，奈米塑膠可能經由破壞緊密連接與影響胞內ROS平衡而導致上皮功能的破壞。研究結果建議，相較於日常使用的塑膠，散佈在環境中的微米和奈米塑膠可能會藉由影響上皮細胞緊密連接與ROS，滲透過上皮屏障，進而對我們的健康產生相當之影響。

The global production of plastics has exceeded 350 million tons[1]; eventually, plastic is degraded into microplastics/nanoplastics and released into the environment. However, the potential health effects of nanoplastics, particularly on epithelial tissues, remain largely unexplored. This study uses a microfluidic system to establish a multi-layer model that reconstructs the epithelial barrier. Our microfluidic chip design incorporated medium-exchange channels and a side-view observation window to enable long-term culture and real-time monitoring.
A multi-layer cell model is established via layer-by-layer co-culture of human keratinocytes and fibroblasts representing the epidermis and dermis; the thickness of the on-chip epidermis(0.1-0.2mm) and the on-chip dermis(0.3-0.6mm) is close to the clinical observations.
Subsequent exposure of the epithelial barrier to nanoplastics resulted in the disassembly of tight junctions along intercellular contacts, indicating compromised barrier integrity. Furthermore, nanoplastics were found to deeply penetrate the multicellular structure, posing a significant challenge to the barrier's function.
Moreover, we observed an increase in intracellular reactive oxygen species (ROS), suggesting the potential induction of mitochondrial dysfunction upon internalization of nanoplastics. These findings indicate that nanoplastic exposure can disrupt epithelial functions by perturbing tight junction assembly and compromising mitochondrial integrity. Considering the critical roles of tight junctions and ROS in maintaining homeostasis and disease progression, our findings suggest an alternative impact of nanoplastic on our health by interrupting the junction assembly.

Abstract i
中文摘要 ii
誌謝 iii
目錄 iv
圖目錄 vi
第一章、導論 1
1.1、研究動機 1
1.2、文獻回顧 2
1.2.1、體外培養模型背景介紹 2
1.2.1.1、體外培養模型之重要性及應用 2
1.2.2、體外三維上皮組織模型 5
1.2.2.1 、體外三維上皮組織模型組成與培養介紹 5
1.2.3、體外上皮組織模型之功能性---緊密連接之重要影響 7
1.2.3.1 、緊密連接介紹 7
1.2.3.2 、上皮組織模型之功能性與緊密連接之關聯[28] 7
1.2.4、評估體外上皮組織模型暴露於塑膠微粒下之影響 9
1.2.4.1 、塑膠微粒之介紹 9
1.2.4.2 、微塑膠粒和奈米塑膠粒對人類健康的潛在毒性影響 10
1.2.4.3 、研究奈米顆粒暴露之體外模型介紹 11
1.3、研究主題 & 實驗流程 14
2.1、實驗藥品 & 器材 15
2.2、微流道裝置設計與製造 16
2.3、細胞培養 20
2.4、免疫螢光染色 21
2.5、細胞氧化壓力(OS)定量定量 22
第三章、研究結果與討論 23
3.1、微流道系統建立三維上皮組織模型 23
3.1.1、微流道裝置的運作 23
3.1.2、基底濃度對於成纖維細胞層生長狀況之影響 23
3.1.3、微流道裝置細胞培養 25
3.1.4、三維上皮組織模型之緊密連接分布 27
3.2、上皮組織模型暴露於奈米塑膠環境下之影響 29
3.2.1、奈米塑膠之表面修飾和濃度對於物質滲透入上皮組織模型的影響 29
3.2.2、奈米塑膠之表面修飾和濃度對於ROS生成之影響 32
3.2.3、奈米塑膠之表面修飾和濃度對於細胞緊密連接之影響 34
3.2.4、奈米塑膠之表面修飾和濃度對於細胞型態之影響 36
第四章、結論與未來展望 38
參考文獻 40

1. Alimba, C.G. and C. Faggio, Microplastics in the marine environment: Current trends in environmental pollution and mechanisms of toxicological profile. Environmental Toxicology and Pharmacology, 2019. 68: p. 61-74.
2. Bedard, P., et al., Innovative Human Three-Dimensional Tissue-Engineered Models as an Alternative to Animal Testing. Bioengineering-Basel, 2020. 7(3).
3. Ronaldson-Bouchard, K. and G. Vunjak-Novakovic, Organs-on-a-Chip: A Fast Track for Engineered Human Tissues in Drug Development. Cell Stem Cell, 2018. 22(3): p. 310-324.
4. Brancato, V., et al., Could 3D models of cancer enhance drug screening? Biomaterials, 2020. 232.
5. Antoni, D., et al., Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci, 2015. 16(3): p. 5517-27.
6. Li, X.J., et al., Microfluidic 3D cell culture: potential application for tissue-based bioassays. Bioanalysis, 2012. 4(12): p. 1509-25.
7. Jensen, C. and Y. Teng, Is It Time to Start Transitioning From 2D to 3D Cell Culture? Front Mol Biosci, 2020. 7: p. 33.
8. Salinas-Vera, Y.M., et al., Three-Dimensional 3D Culture Models in Gynecological and Breast Cancer Research. Front Oncol, 2022. 12: p. 826113.
9. Jensen, C. and Y. Teng, Is It Time to Start Transitioning From 2D to 3D Cell Culture? Frontiers in Molecular Biosciences, 2020. 7.
10. van Duinen, V., et al., Microfluidic 3D cell culture: from tools to tissue models. Curr Opin Biotechnol, 2015. 35: p. 118-26.
11. Limongi, T., et al., Microfluidics for 3D Cell and Tissue Cultures: Microfabricative and Ethical Aspects Updates. Cells, 2022. 11(10).
12. Langhans, S.A., Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning. Front Pharmacol, 2018. 9: p. 6.
13. Olaniyi, R., et al., Staphylococcus aureus-Associated Skin and Soft Tissue Infections: Anatomical Localization, Epidemiology, Therapy and Potential Prophylaxis. Curr Top Microbiol Immunol, 2017. 409: p. 199-227.
14. Baroni, A., et al., Structure and function of the epidermis related to barrier properties. Clin Dermatol, 2012. 30(3): p. 257-62.
15. Hendriks, A., et al., Human Organotypic Models for Anti-infective Research. Curr Top Microbiol Immunol, 2021. 430: p. 77-99.
16. Yousef, H., M. Alhajj, and S. Sharma, Anatomy, Skin (Integument), Epidermis, in StatPearls. 2022: Treasure Island (FL).
17. Yuki, T., et al., Impaired Tight Junctions in Atopic Dermatitis Skin and in a Skin-Equivalent Model Treated with Interleukin-17. Plos One, 2016. 11(9).
18. Kuo, W.T., et al., Tight junction proteins occludin and ZO-1 as regulators of epithelial proliferation and survival. Annals of the New York Academy of Sciences, 2022. 1514(1): p. 21-33.
19. Randall, M.J., et al., Advances in the Biofabrication of 3D Skin in vitro: Healthy and Pathological Models. Front Bioeng Biotechnol, 2018. 6: p. 154.
20. Bogdanowicz, D.R. and H.H. Lu, Studying cell-cell communication in co-culture. Biotechnol J, 2013. 8(4): p. 395-6.
21. Rousi, E., et al., An innervated skin 3D in vitro model for dermatological research. In vitro models, 2022.
22. Stevenson, B.R., et al., Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J Cell Biol, 1986. 103(3): p. 755-66.
23. Furuse, M., et al., Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol, 2002. 156(6): p. 1099-111.
24. Brandner, J.M., Importance of Tight Junctions in Relation to Skin Barrier Function. Curr Probl Dermatol, 2016. 49: p. 27-37.
25. Katsarou, S., et al., The Role of Tight Junctions in Atopic Dermatitis: A Systematic Review. J Clin Med, 2023. 12(4).
26. Groschwitz, K.R. and S.P. Hogan, Intestinal barrier function: molecular regulation and disease pathogenesis. J Allergy Clin Immunol, 2009. 124(1): p. 3-20; quiz 21-2.
27. Varadarajan, S., R.E. Stephenson, and A.L. Miller, Multiscale dynamics of tight junction remodeling. J Cell Sci, 2019. 132(22).
28. Murakami, M., et al., Observation of a tight junction structure generated in LbL-3D skin reconstructed by layer-by-layer cell coating technique. J Tissue Eng Regen Med, 2021. 15(9): p. 798-803.
29. Miyazaki, H., et al., A novel strategy to engineer pre-vascularized 3-dimensional skin substitutes to achieve efficient, functional engraftment. Sci Rep, 2019. 9(1): p. 7797.
30. Basler, K., et al., The role of tight junctions in skin barrier function and dermal absorption. J Control Release, 2016. 242: p. 105-118.
31. Yoshida, K., et al., Functional tight junction barrier localizes in the second layer of the stratum granulosum of human epidermis. J Dermatol Sci, 2013. 71(2): p. 89-99.
32. Luciano, M., et al., Appreciating the role of cell shape changes in the mechanobiology of epithelial tissues. Biophysics Reviews, 2022. 3(1).
33. Lai, H., X. Liu, and M. Qu, Nanoplastics and Human Health: Hazard Identification and Biointerface. Nanomaterials (Basel), 2022. 12(8).
34. Schroter, L. and N. Ventura, Nanoplastic Toxicity: Insights and Challenges from Experimental Model Systems. Small, 2022. 18(31): p. e2201680.
35. Yee, M.S., et al., Impact of Microplastics and Nanoplastics on Human Health. Nanomaterials (Basel), 2021. 11(2).
36. Lin, S., et al., Metabolomics Reveal Nanoplastic-Induced Mitochondrial Damage in Human Liver and Lung Cells. Environ Sci Technol, 2022. 56(17): p. 12483-12493.
37. Zhang, X., G.H. Ma, and W. Wei, Simulation of nanoparticles interacting with a cell membrane: probing the structural basis and potential biomedical application. Npg Asia Materials, 2021. 13(1).
38. Lett, Z., et al., Environmental microplastic and nanoplastic: Exposure routes and effects on coagulation and the cardiovascular system. Environ Pollut, 2021. 291: p. 118190.
39. Lundqvist, M., et al., Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proceedings of the National Academy of Sciences of the United States of America, 2008. 105(38): p. 14265-14270.
40. Garcia-Alvarez, R. and M. Vallet-Regi, Hard and Soft Protein Corona of Nanomaterials: Analysis and Relevance. Nanomaterials, 2021. 11(4).
41. Cao, J.Y., et al., Coronas of micro/nano plastics: a key determinant in their risk assessments. Particle and Fibre Toxicology, 2022. 19(1).
42. Enfrin, M., et al., Release of hazardous nanoplastic contaminants due to microplastics fragmentation under shear stress forces. Journal of Hazardous Materials, 2020. 384.
43. Qiao, J.Y., et al., Perturbation of gut microbiota plays an important role in micro/nanoplastics-induced gut barrier dysfunction. Nanoscale, 2021. 13(19): p. 8806-8816.
44. Hwang, J., et al., Potential toxicity of polystyrene microplastic particles. Sci Rep, 2020. 10(1): p. 7391.
45. Leslie, H.A., et al., Discovery and quantification of plastic particle pollution in human blood. Environ Int, 2022. 163: p. 107199.
46. Malatesta, M., Transmission Electron Microscopy as a Powerful Tool to Investigate the Interaction of Nanoparticles with Subcellular Structures. International Journal of Molecular Sciences, 2021. 22(23).
47. Gamboa, J.M. and K.W. Leong, In vitro and in vivo models for the study of oral delivery of nanoparticles. Adv Drug Deliv Rev, 2013. 65(6): p. 800-10.
48. Carton, F. and M. Malatesta, In Vitro Models of Biological Barriers for Nanomedical Research. Int J Mol Sci, 2022. 23(16).
49. Giusti, S., et al., A novel dual-flow bioreactor simulates increased fluorescein permeability in epithelial tissue barriers. Biotechnol J, 2014. 9(9): p. 1175-84.
50. Esch, E.W., A. Bahinski, and D. Huh, Organs-on-chips at the frontiers of drug discovery. Nature Reviews Drug Discovery, 2015. 14(4): p. 248-260.
51. Blank, F., et al., Macrophages and dendritic cells express tight junction proteins and exchange particles in an in vitro model of the human airway wall. Immunobiology, 2011. 216(1-2): p. 86-95.
52. Kik, K., B. Bukowska, and P. Sicinska, Polystyrene nanoparticles: Sources, occurrence in the environment, distribution in tissues, accumulation and toxicity to various organisms. Environmental Pollution, 2020. 262.
53. Zoio, P. and A. Oliva, Skin-on-a-Chip Technology: Microengineering Physiologically Relevant In Vitro Skin Models. Pharmaceutics, 2022. 14(3).
54. Frantz, C., K.M. Stewart, and V.M. Weaver, The extracellular matrix at a glance. J Cell Sci, 2010. 123(Pt 24): p. 4195-200.
55. Schneider, S., et al., Membrane integration into PDMS-free microfluidic platforms for organ-on-chip and analytical chemistry applications. Lab Chip, 2021. 21(10): p. 1866-1885.
56. Abd, E., et al., Skin models for the testing of transdermal drugs. Clin Pharmacol, 2016. 8: p. 163-176.
57. Josan, C., S. Kakar, and S. Raha, Matrigel(R) enhances 3T3-L1 cell differentiation. Adipocyte, 2021. 10(1): p. 361-377.
58. Aisenbrey, E.A. and W.L. Murphy, Synthetic alternatives to Matrigel. Nat Rev Mater, 2020. 5(7): p. 539-551.
59. Lee, S.H., et al., Hydrogel-based three-dimensional cell culture for organ-on-a-chip applications. Biotechnol Prog, 2017. 33(3): p. 580-589.
60. Guillot, C. and T. Lecuit, Mechanics of epithelial tissue homeostasis and morphogenesis. Science, 2013. 340(6137): p. 1185-9.
61. Kirschner, N. and J.M. Brandner, Barriers and more: functions of tight junction proteins in the skin. Ann N Y Acad Sci, 2012. 1257: p. 158-66.
62. Yuki, T., et al., Characterization of Tight Junctions and Their Disruption by UVB in Human Epidermis and Cultured Keratinocytes. Journal of Investigative Dermatology, 2011. 131(3): p. 744-752.
63. Shi, Y., et al., No tight junctions in tight junction protein-1 expressing HeLa and fibroblast cells. Int J Physiol Pathophysiol Pharmacol, 2020. 12(2): p. 70-78.
64. Derr, K., et al., Fully Three-Dimensional Bioprinted Skin Equivalent Constructs with Validated Morphology and Barrier Function. Tissue Eng Part C Methods, 2019. 25(6): p. 334-343.
65. Kumamoto, J., et al., Mathematical-model-guided development of full-thickness epidermal equivalent. Sci Rep, 2018. 8(1): p. 17999.
66. Chen, J., et al., Cellular absorption of polystyrene nanoplastics with different surface functionalization and the toxicity to RAW264.7 macrophage cells. Ecotoxicol Environ Saf, 2023. 252: p. 114574.
67. Roshanzadeh, A., et al., Exposure to nanoplastics impairs collective contractility of neonatal cardiomyocytes under electrical synchronization. Biomaterials, 2021. 278: p. 121175.
68. Mittal, M., et al., Reactive Oxygen Species in Inflammation and Tissue Injury. Antioxidants & Redox Signaling, 2014. 20(7): p. 1126-1167.
69. Blaser, H., et al., TNF and ROS Crosstalk in Inflammation. Trends Cell Biol, 2016. 26(4): p. 249-261.
70. Lan, T., L. Chen, and X. Wei, Inflammatory Cytokines in Cancer: Comprehensive Understanding and Clinical Progress in Gene Therapy. Cells, 2021. 10(1): p. 100.
71. Sun, Z., et al., Exposure to nanoplastics induces mitochondrial impairment and cytomembrane destruction in Leydig cells. Ecotoxicol Environ Saf, 2023. 255: p. 114796.
72. He, Y., et al., Polystyrene nanoplastics deteriorate LPS-modulated duodenal permeability and inflammation in mice via ROS drived-NF-kappaB/NLRP3 pathway. Chemosphere, 2022. 307(Pt 1): p. 135662.