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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78819完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳敏慧 | zh_TW |
| dc.contributor.advisor | Min-Huey Chen | en |
| dc.contributor.author | 游善淳 | zh_TW |
| dc.contributor.author | Shan-Chun Yu | en |
| dc.date.accessioned | 2021-07-11T15:21:42Z | - |
| dc.date.available | 2024-08-16 | - |
| dc.date.copyright | 2019-03-11 | - |
| dc.date.issued | 2019 | - |
| dc.date.submitted | 2002-01-01 | - |
| dc.identifier.citation | 1. Graw, J., The genetic and molecular basis of congenital eye defects. Nat Rev Genet, 2003. 4(11): p. 876-88.
2. Leach, L.L. and D.O. Clegg, Concise Review: Making Stem Cells Retinal: Methods for Deriving Retinal Pigment Epithelium and Implications for Patients With Ocular Disease. Stem Cells, 2015. 33(8): p. 2363-73. 3. Lim, J.Y., et al., Brain-derived neurotrophic factor stimulates the neural differentiation of human umbilical cord blood-derived mesenchymal stem cells and survival of differentiated cells through MAPK/ERK and PI3K/Akt-dependent signaling pathways. J Neurosci Res, 2008. 86(10): p. 2168-78. 4. Sommerfeld, M.T., Schweigreiter, R., Barde, Y. A., & Hoppe, E., Down-regulation of the Neurotrophin Receptor TrkB following Ligand Binding EVIDENCE FOR AN INVOLVEMENT OF THE PROTEASOME AND DIFFERENTIAL REGULATION OF TrkA AND TrkB. Journal of Biological Chemistry, 2000. 275(12): p. 8982-8990. 5. Nazari, H., et al., Stem cell based therapies for age-related macular degeneration: The promises and the challenges. Prog Retin Eye Res, 2015. 48: p. 1-39. 6. Mead, B., et al., Concise Review: Dental Pulp Stem Cells: A Novel Cell Therapy for Retinal and Central Nervous System Repair. Stem Cells, 2017. 35(1): p. 61-67. 7. Perry, B.C., et al., Collection, cryopreservation, and characterization of human dental pulp-derived mesenchymal stem cells for banking and clinical use. Tissue Eng Part C Methods, 2008. 14(2): p. 149-56. 8. Mead, B., et al., Intravitreally transplanted dental pulp stem cells promote neuroprotection and axon regeneration of retinal ganglion cells after optic nerve injury. Invest Ophthalmol Vis Sci, 2013. 54(12): p. 7544-56. 9. Kim, S.G., et al., Effects of growth factors on dental stem/progenitor cells. Dent Clin North Am, 2012. 56(3): p. 563-75. 10. Sango, K., et al., Myelination in coculture of established neuronal and Schwann cell lines. Histochem Cell Biol, 2012. 137(6): p. 829-39. 11. Duan, P., et al., Human bone marrow stromal cells can differentiate to a retinal pigment epithelial phenotype when co-cultured with pig retinal pigment epithelium using a transwell system. Cell Physiol Biochem, 2013. 31(4-5): p. 601-13. 12. Zhang, X., et al., Coculture of mesenchymal stem cells and endothelial cells enhances host tissue integration and epidermis maturation through AKT activation in gelatin methacryloyl hydrogel-based skin model. Acta Biomater, 2017. 59: p. 317-326. 13. Mathivanan, I., et al., Retinal differentiation of human bone marrow-derived stem cells by co-culture with retinal pigment epithelium in vitro. Exp Cell Res, 2015. 333(1): p. 11-20. 14. Amirpour, N., Nasr-Esfahani, M. H., Esfandiari, E., Razavi, S., & Karamali, F. , Comparing three methods of co-culture of retinal pigment epithelium with progenitor cells derived human embryonic stem cells. International journal of preventive medicine, 2013. 4(11): p. 1243. 15. Michalczyk, K. and M. Ziman, Nestin structure and predicted function in cellular cytoskeletal organisation. Histol Histopathol, 2005. 20(2): p. 665-71. 16. Guo, J., C. Walss-Bass, and R.F. Luduena, The beta isotypes of tubulin in neuronal differentiation. Cytoskeleton (Hoboken), 2010. 67(7): p. 431-41. 17. Bonnamain, V., et al., Human dental pulp stem cells cultured in serum-free supplemented medium. Front Physiol, 2013. 4: p. 357. 18. Shi, S., and Stan Gronthos, Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. Journal of bone and mineral research, (2003):. 18.4 p. 696-704. 19. He, J., et al., CD90 is identified as a candidate marker for cancer stem cells in primary high-grade gliomas using tissue microarrays. Mol Cell Proteomics, 2012. 11(6): p. M111 010744. 20. Ashery-Padan, R., Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye. Genes & Development, 2000. 14(21): p. 2701-2711. 21. Zhang, X., et al., Pax6 is a human neuroectoderm cell fate determinant. Cell Stem Cell, 2010. 7(1): p. 90-100. 22. Zuber, M.E., et al., Specification of the vertebrate eye by a network of eye field transcription factors. Development, 2003. 130(21): p. 5155-67. 23. Van Heyningen, V., and Kathleen A. Williamson. , PAX6 in sensory development. Human molecular genetics, 2002. 11.10 p. 1161-1167. 24. Fresno Vara, J.A., et al., PI3K/Akt signalling pathway and cancer. Cancer Treat Rev, 2004. 30(2): p. 193-204. 25. Rahmani, A., et al., Neurogenesis and increase in differentiated neural cell survival via phosphorylation of Akt1 after fluoxetine treatment of stem cells. Biomed Res Int, 2013. 2013: p. 582526. 26. Zhou, J., et al., Enhanced functional properties of corneal epithelial cells by coculture with embryonic stem cells via the integrin beta1-FAK-PI3K/Akt pathway. Int J Biochem Cell Biol, 2011. 43(8): p. 1168-77. 27. Maya Schuldiner , R.E., Amir Eden , Ofra Yanuka , Joseph Itskovitz-Eldor , and N.B. Ronald S. Goldsteinc, Induced neuronal differentiation of human embryonic stem cells. Brain research, 2001(913(2)): p. 201-205. 28. Lee, G., et al., Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat Biotechnol, 2007. 25(12): p. 1468-75. 29. Mellough, C.B., et al., Efficient stage-specific differentiation of human pluripotent stem cells toward retinal photoreceptor cells. Stem Cells, 2012. 30(4): p. 673-86. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78819 | - |
| dc.description.abstract | 視網膜對於視覺而言是關鍵構造，視網膜的受損、疾病、退化皆有可能造成視覺方面的障礙。幹細胞療法相較於過去的治療方式，例如支持性療法及神經營養因子（neurotrophins）輔助療法，也許會是更有潛力的治療方式。過去的研究中曾經提及，幹細胞對於視網膜細胞以及視神經有修復與再生的能力，對於保護神經免於傷害以及促進神經修復方面亦有功效，並且相較於人類胚胎幹細胞或是骨髓幹細胞，人類牙髓幹細胞（dental pulp stem cells, DPSCs）取得容易、在倫理上的爭議較小，並且具有分化為脂肪細胞、軟骨細胞、肌細胞等多種細胞的能力，說明了牙髓幹細胞是作為幹細胞治療的合適來源。
人類視網膜色素上皮細胞（retinal pigmented epithelium, RPE）位於視網膜外皮，在胚胎發育時參與了眼球形成的過程。神經生長因子（NGF, nerve growth factor）是一種同二聚體肽類，能夠調節細胞的生長以及促進神經分化。本研究之假說為，將人類牙髓幹細胞以神經生長因子誘導處理後，初步分化為神經前驅細胞，再與人類視網膜色素上皮細胞共同培養後，能夠表現出視網膜細胞標記，以螢光染色以及收集蛋白質測試Akt及其磷酸化型態的存在，並且加入Akt-PI3K 之抑制劑LY294002對共同培養之細胞進行前處理，來探討牙髓幹細胞的階段性分化是否與Akt生化路徑相關。 細胞免疫螢光染色的結果顯示，加入神經生長因子進行初步誘導分化的組別表現Pax6較未誘導的組別早，細胞形態方面亦有明顯差異；而西方墨點法的結果顯示，共同培養後所收集的牙髓幹細胞之蛋白質表現，在加入抑制劑LY294002的組別中，磷酸化Akt顯著地減少。本實驗的結論為人類牙髓幹細胞經過神經生長因子處理後，與視網膜色素上皮細胞共同培養，可以促進細胞分化而表現出Pax6視網膜標記，而磷酸化akt的表現在細胞間接接觸之後增加，加入抑制劑則使磷酸化akt顯著減少。 | zh_TW |
| dc.description.abstract | One of the key structures of human vision forming is retina. Visual impairment could be caused by damage, regression and disease of retina. Compared with previous therapies such as traditional supportive treatment or neurotrophins injection, stem cell therapy is a more promising approach.
Stem cells are referred to be able to repair and regenerate the optic nerves and retinal cells and they are also effective in protecting nerves from damage in the past researches. Human dental pulp stem cells are more accessible stem cell source comparing to human embryo stem cells or bone marrow stem cells with less ethical concerns, presenting differential ability to multiple cells including adipose cells, chondrocytes and muscle cells. Retinal pigmented epithelium cells (RPE) which located in retinal epithelium is highly involved in eye development. NGF is able to regulate cell growth and facilitate neural differentiation. We hypothesized that DPSC-derived neural progenitors would differentiate to express retinal marker Pax6 after coculturing with RPE in vitro and is related to akt pathway, which were evaluated by immunocytochemistry and protein collection. Our immunocytochemistry and Western blotting results indicated that NGF-primed group required less time to express Pax6, and pakt level is reduced significantly in inhibitor LY294002 group. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T15:21:42Z (GMT). No. of bitstreams: 1 ntu-108-R05422015-1.pdf: 5124551 bytes, checksum: af55f32b86870e3d5dc35d43c28e3e93 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 口試委員會審定書………………………… ………………. …………………………………………………#
誌謝…………………………………………………………. ………………. ………………. ……………………. i 中文摘要…………………………………………………………………………. ………………. ……………… ii ABSTRACT…………………………………………………………………………. iii CONTENTS…………………………………………………………………………. iv LIST OF FIGURES…………………………………………………………………. vii LIST OF TABLES…………………………………………………………………. x Chapter 1 前言………………………………………………………… 1 1.1 眼球之基本構造（Basic Structure Of Eyeball）……………………………………….. 2 1.2 視網膜的退化（Retinal Degeneration）………………………. ………………………….. 3 1.3 視網膜退化的治療（Therapeutic Approach for Retinal Degeneration）…. 3 1.4 人類牙髓幹細胞（dental pulp stem cell）……………………………………………….. 5 1.5 神經分化與神經前驅細胞（neural differentiation and neural progenitor cells）…………………………………………………………………………………………………………… 6 1.6 表現視網膜標記之分化（Cell differentiation to express retinal marker）…………………………………………………………………………………………………………… 7 1.7 細胞標記（Cell Markers）……………………………………………………………………….. 8 1.7.1 神經細胞標記（Neurala Markers）………………………………………….. 8 1.7.2 幹細胞標記（Stemness markers）………………… ……………………….. 8 1.7.3 視網膜細胞標記（Retinal Markers）……………………………………….. 9 1.8 AKT生化路徑 …………………………………………………………..………………………….. 9 Chapter 2 實驗假說與目的……………………………………………………………………. 10 Chapter 3 實驗方法與材料………………………………………………………………….. 11 3. 1 實驗設計（Experiment Design）……………………………………………………………… 11 3. 2 人類牙髓幹細胞培養（Culturing of DPSCs）………………………………………15 3. 3 視網膜色素上皮細胞培養(Culturing of RPE)……………………………………16 3. 4 初步神經分化誘導方式（Initial Neural Differentiation Method）………………………………………………………………………………………………………16 3. 5視網膜分化誘導方式（Retinal Differentiation Method）………………………………………………………………………………………………………17 3. 6免疫螢光染色 （Immunocytochemistry Staining）………………………………17 3.6.1 細胞化學染色（Immunocytochemistry Staining）…………………17 3.7 西方墨點分析法 (Western blot)…………………………………………………………18 3.7.1 蛋白質的萃取及定量（Protein Extraction）…………………………18 3.7.2 SDS聚丙烯醯胺膠電泳法分析（SDS-PAGE）……………………………19 3.7.3 蛋白質轉移（protein transfter）……………………………………………20 3.7.4 免疫反應（immunoreaction）……………………………………………………20 3.7.5 化學冷光呈色（chemiluminescence）………………………………………20 3.7.6 統計分析（statistical analysis）………………………………………..21 Chapter 4 實驗結果………………………………………….………………………………….. 21 4. 1 人類牙髓幹細胞(DPSCs)之型態觀察及檢驗……………………………………21 4. 2 視網膜上皮色素細胞(RPE)培養之型態觀察及檢驗……………………21 4. 3 免疫螢光染色的細胞標記之檢驗(Positive Controls for Cell Markers) …22 4.4 蛋白質收集及西方墨點法之檢驗（Protein Extraction and Western Blot）………………………………………………………………………………………………………………22 4. 5 初步研究(preliminary Studies) ……………………………………………………22 4. 5.1誘導分化之培養基選擇（Selection of Induction Medium）………………………………………………………………………………………………22 4. 5.2利用神經生長因子使牙髓幹細胞初步分化（Initial Neural Differentiation by NGF Priming）…………………………………………………23 4. 5.3共同培養之設計（Co-culture Assay Design）………………………… 23 4.6 牙髓幹細胞分化為神經前驅細胞（DPSCs Differentiate into Neural Progenitor Cells）………………………………………………………………24 4.7 牙髓幹細胞分化之神經前驅細胞繼續分化為視網膜標記表現細胞（Neural Progenitor Cells Differentiate into Retinal Marker Expressing Cells）…………………………………………………………………………25 4.8 分化過程中Akt以及p-akt的表現(akt and pakt expression during differentiation)……………………………………………………………………………26 Chapter 5 討論………………………………………………………………….……………………. 27 5. 1 比較幹細胞的視網膜分化結果之差異（Comparison of Stem Cells’Retinal differentiation）……………………………………………………………………………………………27 5. 2 比較神經生長因子對於牙髓幹細胞分化之影響………………………………… 27 5. 3 比較直接及間接之共同培養方式（comparison between Direct and Indirect Co-culture Assay）………………………………………………………………… 28 5. 4 Akt生化路徑於此Pax6表現細胞分化中的參與（Involving of Akt pathway in differentiation）………………………………………………………………… 29 Chapter 6 結論………………………………………………………………….……………….……30 Chapter 7 未來研究方向……………………………………………………………………….30 REFERENCE………………………………………………………………………………………….…….……61 | - |
| dc.language.iso | zh_TW | - |
| dc.subject | 牙髓幹細胞 | zh_TW |
| dc.subject | 共同培養 | zh_TW |
| dc.subject | 視網膜色素上皮細胞 | zh_TW |
| dc.subject | akt | zh_TW |
| dc.subject | co-culture | en |
| dc.subject | RPE | en |
| dc.subject | DPSCs | en |
| dc.subject | akt | en |
| dc.title | 人類牙髓幹細胞藉由與視網膜色素上皮細胞共同培養分化為Pax6表現細胞 | zh_TW |
| dc.title | Differentiation of Dental Pulp Stem Cells into Pax6 Expression Cells after coculturing with RPE Cells | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 107-1 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 陳偉勵;周涵怡;陳容慈 | zh_TW |
| dc.contributor.oralexamcommittee | Wei-Li Chen;Han-Yi Chou;Jung-Tsu Chen | en |
| dc.subject.keyword | 牙髓幹細胞,視網膜色素上皮細胞,共同培養,akt, | zh_TW |
| dc.subject.keyword | DPSCs,RPE,co-culture,akt, | en |
| dc.relation.page | 63 | - |
| dc.identifier.doi | 10.6342/NTU201900314 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2019-02-13 | - |
| dc.contributor.author-college | 醫學院 | - |
| dc.contributor.author-dept | 臨床牙醫學研究所 | - |
| dc.date.embargo-lift | 2024-03-11 | - |
| 顯示於系所單位： | 臨床牙醫學研究所 | |
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