( 您好!臺灣時間:2021/03/05 08:18
字體大小: 字級放大   字級縮小   預設字形  


研究生(外文):Huei-Wen Cheng
論文名稱(外文):Promoting the hair-inductive activity of keratinocytes by co-culturing with dermal papilla cells and form 3D dermal papilla microtissues for hair follicle reconstruction
  • 被引用被引用:0
  • 點閱點閱:35
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0

因此本研究的目的在於提供一種簡易的方法用來製備及培養真皮乳頭細胞和表皮細胞形成的微球組織,有可以達到高產量、大小均一的特點,且觀察其是否能在生物體內的環境下達到毛髮新生的效果。本研究選用聚二甲基矽氧烷(Polydimethylsiloxane, PDMS)作為材料,由於其本身良好的生物相容性及細胞不易貼附的特性,將真皮乳頭細胞培養在已塗覆PDMS的96孔盤內便能使其自行聚集形成微球組織,再加上表皮細胞在外層包覆並透過免疫螢光染色確保其仍保有促進毛囊新生能力。為了提供適當的生長骨架,以膠原蛋白(collagen type I)與細胞微球組織混合使膠原蛋白包覆著微球成膠,並植入裸鼠體內觀察其發育形態,以證明本實驗設計之真皮乳頭細胞微球能促進毛囊的生成及毛髮再生,並在未來有應用於毛囊重建之組織工程的潛力。
Alopecia is a disturbing problem effecting men and women of all ages. Because the syndrome directly affects the appearance, cause physiological impacts on patient. The treatment for alopecia involves either hair transplant, pharmacotherapy or phototherapy. But these treatment options are fraught with problems of cost, side effects, and treatment effect is unstable. Recently, with the development and trend of tissue engineering, more and more scholars study on hair follicle regeneration. Cell-based therapies have focused on the dermal papilla cell. However, many obstacles exist, inducing ability and maintenance of dermal papilla productivity after several passages of culture. The natural hair development process depends on a series of interactions between dermal papilla cells and epidermal cells. Thus, the DP cells and epidermal cells are cultured together to form a three-dimensional spheroid structure for promote cells interaction and retain the ability to induce hair regeneration.

Therefore, the aim of this study was to culture dermal papilla cells and epidermal cells to form microsphere tissues, which are high-throughput and can be produced in uniform-size manner, and to investigate whether these microsphere tissues could induce hair regeneration. Polydimethylsiloxane (PDMS) was selected as the material. Due to its good biocompatibility and low cell adhesion, dermal papilla cells were cultured in 96-well discs coated with PDMS to form micro spheroids. Moreover, epidermal cells were added to form “core-shell” structure, and immunofluorescence staining was used to ensure that they still retained the ability to promote hair follicle regeneration. On the other hand, in order to provide an appropriate scaffold, we choose collagen type I as biomaterial scaffolds. Microspheres were collected and mixed with collagen solution to form a hydrogel and transplanted into nude mice, then observe their morphology to prove that this experimental design can induce hair regeneration and has potential application in hair follicle reconstruction in the future.
致謝 I
摘要 II
目錄 V
圖目錄 VII
第一章 序論 1
1.1毛髮與毛囊 1
1.2 毛囊生長週期 2
1.3禿髮 3
1.4禿髮治療手段 3
1.4.1 藥物治療 3
1.4.2 光治療 4
1.4.3 自體頭髮移植 4
1.5 毛囊組織工程--毛囊重建 4
1.5.1 毛囊幹細胞 4
1.5.2 三維細胞培養(3D culture) 5
1.5.3 共培養系統(co-culture) 5
1.6 動物實驗模型(IN VIVO) 6
第二章 研究概述 8
2.1 研究背景 8
2.2 研究動機與目的 8
2.3 方法簡述 9
2.4 實驗流程圖 9
第三章 實驗材料與方法 10
3.1 實驗藥品 10
3.2 實驗儀器 12
3.3 低貼附性96-WELL之製備 13
3.4 人類毛囊真皮乳頭細胞及角質形成細胞之培養 13
3.5 新生小鼠角質細胞之分離與培養 14
3.6 角質形成細胞KERATINOCYTE與真皮乳頭細胞(DP CELL)共培養系統 14
3.7 CORE-SHELL細胞培養 15
3.8 PKH26細胞紅螢光染色 16
3.9 PKH67細胞綠螢光染色 16
3.10 細胞生存力染色 16
3.11 免疫細胞化學(IMMUNOCYTOCHEMISTRY, ICC)與鹼性磷酸酶染色 17
3.14 組織切片染色 20
第四章 結果與討論 21
4.1 新生小鼠角質細胞的培養及形態觀察 21
4.2 人類真皮乳頭細胞培養至細胞低貼附性之96孔盤的形態觀察 22
4.3 真皮乳頭細胞與角質細胞共同成球 23
4.4 真皮乳頭細胞微球組織活性分析 25
4.5 IN VITRO培養下真皮乳頭誘導毛囊再生之能力 26
4.6 真皮乳頭微球組織在活體內誘導毛囊生成之能力驗證 30
第五章 結論 32
第六章 參考文獻 33
[1]Driskell, R.R., C. Clavel, M. Rendl, and F.M. Watt, Hair follicle dermal papilla cells at a glance. J Cell Sci, 2011. 124(8): p. 1179-1182.
[2]Rishikaysh, P., K. Dev, D. Diaz, W. Qureshi, S. Filip, and J. Mokry, Signaling involved in hair follicle morphogenesis and development. International journal of molecular sciences, 2014. 15(1): p. 1647-1670.
[3]Stenn, K. and R. Paus, Controls of hair follicle cycling. Physiological reviews, 2001. 81(1): p. 449-494.
[4]Filbrandt, R., N. Rufaut, L. Jones, and R. Sinclair, Primary cicatricial alopecia: diagnosis and treatment. Cmaj, 2013. 185(18): p. 1579-1585.
[5]Avci, P., G.K. Gupta, J. Clark, N. Wikonkal, and M.R. Hamblin, Low‐level laser (light) therapy (LLLT) for treatment of hair loss. Lasers in surgery and medicine, 2014. 46(2): p. 144-151.
[6]Yoo, B.-Y., Y.-H. Shin, H.-H. Yoon, Y.-K. Seo, and J.-K. Park, Hair multiplication with dermal papilla like tissue containing human dermal papilla cells. Biotechnology and bioprocess engineering, 2014. 19(3): p. 394-403.
[7]Rendl, M., L. Lewis, and E. Fuchs, Molecular dissection of mesenchymal–epithelial interactions in the hair follicle. PLoS biology, 2005. 3(11): p. e331.
[8]Jahoda, C., K. Horne, and R. Oliver, Induction of hair growth by implantation of cultured dermal papilla cells. Nature, 1984. 311(5986): p. 560.
[9]Yang, C.-C. and G. Cotsarelis, Review of hair follicle dermal cells. Journal of dermatological science, 2010. 57(1): p. 2-11.
[10]Higgins, C.A., G.D. Richardson, D. Ferdinando, G.E. Westgate, and C.A. Jahoda, Modelling the hair follicle dermal papilla using spheroid cell cultures. Experimental dermatology, 2010. 19(6): p. 546-548.
[11]Hsieh, C.-H., J.-L. Wang, and Y.-Y. Huang, Large-scale cultivation of transplantable dermal papilla cellular aggregates using microfabricated PDMS arrays. Acta biomaterialia, 2011. 7(1): p. 315-324.
[12]Goers, L., P. Freemont, and K.M. Polizzi, Co-culture systems and technologies: taking synthetic biology to the next level. Journal of The Royal Society Interface, 2014. 11(96): p. 20140065.
[13]Inamatsu, M., T. Matsuzaki, H. Iwanari, and K. Yoshizato, Establishment of rat dermal papilla cell lines that sustain the potency to induce hair follicles from afollicular skin. Journal of Investigative Dermatology, 1998. 111(5): p. 767-775.
[14]Bak, S.S., M.H. Kwack, H.S. Shin, J.C. Kim, M.K. Kim, and Y.K. Sung, Restoration of hair-inductive activity of cultured human follicular keratinocytes by co-culturing with dermal papilla cells. Biochemical and biophysical research communications, 2018. 505(2): p. 360-364.
[15]Mahjour, S.B., F. Ghaffarpasand, and H. Wang, Hair follicle regeneration in skin grafts: current concepts and future perspectives. Tissue Engineering Part B: Reviews, 2011. 18(1): p. 15-23.
[16]Zheng, Y., X. Du, W. Wang, M. Boucher, S. Parimoo, and K.S. Stenn, Organogenesis from dissociated cells: generation of mature cycling hair follicles from skin-derived cells. Journal of Investigative Dermatology, 2005. 124(5): p. 867-876.
[17]Ohyama, M., Use of human intra-tissue stem/progenitor cells and induced pluripotent stem cells for hair follicle regeneration. Inflammation and regeneration, 2019. 39(1): p. 4.
[18]Cooley, J., Follicular cell implantation: an update on" hair follicle cloning". Facial plastic surgery clinics of North America, 2004. 12(2): p. 219-224.
[19]Higgins, C.A., J.C. Chen, J.E. Cerise, C.A. Jahoda, and A.M. Christiano, Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth. Proceedings of the National Academy of Sciences, 2013. 110(49): p. 19679-19688.
[20]Tharmalingam, T., H. Ghebeh, T. Wuerz, and M. Butler, Pluronic enhances the robustness and reduces the cell attachment of mammalian cells. Molecular biotechnology, 2008. 39(2): p. 167-177.
[21]Jahoda, C., A.J. Reynolds, C. Chaponnier, J.C. Forester, and G. Gabbiani, Smooth muscle alpha-actin is a marker for hair follicle dermis in vivo and in vitro. Journal of cell science, 1991. 99(3): p. 627-636.
[22]Müller-Röver, S., E.J. Peters, V.A. Botchkarev, A. Panteleyev, and R. Paus, Distinct patterns of NCAM expression are associated with defined stages of murine hair follicle morphogenesis and regression. Journal of Histochemistry & Cytochemistry, 1998. 46(12): p. 1401-1409.
[23]Soma, T., M. Tajima, and J. Kishimoto, Hair cycle-specific expression of versican in human hair follicles. Journal of dermatological science, 2005. 39(3): p. 147-154.
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔