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研究生： 歐陽貝依
Ou Yang, Pei-Yi
論文名稱： 以不同細胞組成3D層片促進周邊神經再生
Using Different Cells to Form 3D Cell Sheets for Peripheral Nerve Regeneration
指導教授： 吳佳慶
Wu, Chia-Ching
學位類別： 碩士
Master
系所名稱： 醫學院 - 細胞生物與解剖學研究所
Institute of Cell Biology and Anatomy
論文出版年： 2025
畢業學年度： 113
語文別： 英文
論文頁數： 126
中文關鍵詞： 周邊神經損傷 、細胞層片 、神經再生 、許旺細胞
外文關鍵詞： Peripheral Nerve Injury (PNI), Cell Sheet, Nerve Regeneration, Schwann Cell (SC)
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    • 周邊神經損傷（Peripheral Nerve Injury, PNI）是一種因壓迫或截斷導致神經功能喪失的損害。本文聚焦於完全截斷型周邊神經損傷的修復治療方法。當神經斷裂距離過大時，由於神經再生能力有限，修復往往失敗，並伴隨神經瘤形成、感覺喪失及肌肉萎縮，這對臨床治療構成重大挑戰。本研究旨在探討不同細胞製成的細胞層片（Cell Sheet）在大鼠周邊神經損傷模型中的應用，並分析其對神經再生及功能恢復的潛力。
    我們製備了多種類型的細胞層片，包括神經來源的層片：許旺細胞（Schwann Cell, SC）、脂肪幹細胞（Adipose Stem Cell, ASC）、以及脂肪幹細胞分化的許旺細胞樣細胞（Adipose Stem Cell-Derived Schwann Cell, ASCSC）；以及肌肉來源的層片：肌母細胞（Myoblast）和肌管細胞（Myotube）。通過qPCR、免疫螢光染色及Western Blot分析這些細胞層片在基因及蛋白層面的改變。此外，我們利用大鼠坐骨神經截斷動物模型，評估各層片在損傷後10天對周邊神經修復的效果，並以步態分析（Gait Analysis）、腓腸肌重量比（Relative Gastrocnemius Muscle Weight, RGMW）、及組織免疫螢光染色作為再生指標。
    目前的研究結果顯示，神經來源的細胞層片（特別是SC和ASCSC）可顯著促進神經再生。在步態分析中，這些細胞層片顯著改善了動物的運動模式，並增加了受損神經支配的肌肉重量。此外，軸突修復長度及再髓鞘化相關蛋白的表現亦有所提升，其修復效果接近健康組（Sham）。相比之下，肌肉來源的層片在功能恢復方面的效果有限，動物的步態異常及肌肉萎縮仍然持續，可能與肌肉層片缺乏支持神經修復的微環境有關。然而，這些結果也間接驗證了我們的假設：肌肉層片雖然抑制了神經功能的進一步修復，但成功減少了損傷部位的過度細胞增生，從而有效避免了神經瘤的形成。
    本研究證實，神經來源的細胞層片（特別是SC和ASCSC）在周邊神經修復中展現了明顯的治療優勢，並強調細胞外基質（Extracellular Matrix, ECM）及培養條件在細胞分化及功能恢復中的關鍵作用。未來研究應進一步探索細胞層片的穩定性、ECM與細胞間的交互機制，以推動細胞層片技術在再生醫學及臨床應用中的進一步發展。

    Peripheral nerve injury (PNI), caused by compression or transection, leads to functional deficits and poses significant challenges in clinical treatment. When the gap between nerve ends is too large, the limited regenerative capacity of nerves often results in repair failure, accompanied by complications such as neuroma formation, sensory loss, and muscle atrophy, making it a formidable obstacle in clinical management. This study explores the potential of using various cell sheets as therapeutic tools for peripheral nerve regeneration in a rat sciatic nerve transection model, analyzing their ability to promote regeneration and functional recovery.
    We prepared neural-derived cell sheets, including Schwann cells (SC), adipose stem cells (ASC), and adipose stem cell-derived Schwann-like cells (ASCSC), as well as muscle-derived cell sheets (Myoblast and Myotube). These cell sheets were characterized by qPCR, immunofluorescence staining, and Western blot to analyze their gene and protein expression changes. The efficacy of these sheets was evaluated in a rat sciatic nerve transection model, with outcomes measured at six weeks post-implantation through gait analysis, relative gastrocnemius muscle weight (RGMW), and tissue immunofluorescence imaging as indicators of regeneration.
    The results demonstrated that neural-derived cell sheets, particularly SC and ASCSC, promoted nerve regeneration. Improvements in gait patterns and the increased weight of target muscles were observed compared to the untreated group. Additionally, enhanced axonal regeneration and increased expression of remyelination-associated proteins brought the repair outcomes closer to those of the healthy (Sham) group. In contrast, muscle-derived cell sheets showed limited efficacy in functional recovery, with persistent gait abnormalities and muscle atrophy. These findings suggest that the lack of a supportive microenvironment for nerve repair may limit their effectiveness.
    This study suggests that neural-derived cell sheets, particularly ASCSC, have significant clinical application advantages in peripheral nerve repair and highlights the roles of cell components in 3D sheet and culture conditions in cell differentiations and functional recovery. Future studies should focus on exploring the long-term stability and mechanical properties of these sheets and the interaction mechanisms between sheet and regenerated tissue to provide more evidence for advancing cell sheet technology in regenerative medicine and clinical applications.

    中文摘要 3 ABSTRACT 5 誌謝 7 TABLE OF CONTENTS 8 LIST OF TABLES 12 LIST OF FIGURES 13 CHAPTER 1. RESEARCH BACKGROUND 16 1-1. NERVE INJURY AND REGENERATION 16 1-1-1. Peripheral nerve injury (PNI) 16 1-1-2 Wallerian Degeneration and axonal regrowth 17 1-1-3 Remyelination 17 1-1-4 Neuroma formation 18 1-2 CURRENT TREATMENTS OF PNI 18 1-2-1 Neurorrhaphy 18 1-2-2 Nerve conduit 19 1-2-3 Autogenous nerve transplantation 19 1-2-4 Regenerative peripheral nerve interface (RPNI) 20 1-2-5 Cell therapy 21 1-3 CELL SHEET 23 1-3-1 Adipose stem cell sheet 24 1-3-2 Neuronal cell sheet 24 1-3-3 Muscular cell sheet 25 1-4 MOTIVATION OF CURRENT STUDY 27 1-5 AIMS 29 CHAPTER 2. MATERIALS AND METHODS 30 2-1 CELL CULTURE 30 2-1-1 ASC sphere formation and induction of SC lineage 31 2-1-2 Myogenesis and myotube formation 32 2-2 CELL SHEET FORMATION 33 2-2-1 ASC sheet 34 2-2-2 ASCSC sheet 35 2-2-3 SC sheet 36 2-2-4 Myoblast (C2C12) sheet 37 2-2-5 Myotube cell 38 2-3 WESTERN BLOT 39 2-3-1 protein sample collecting 39 2-3-2 Sample preparation 39 2-3-3 Protein separation by gel electrophoresis 39 2-3-4 Transfer 40 2-3-5 Blocking 40 2-3-6 Primary and secondary antibody 40 2-4 TOTAL RNA EXTRACTION AND REVERSE TRANSCRIPTION-QUANTITATIVE POLYMERASE CHAIN REACTION (QRT-PCR) 43 2-4-1 RNA extraction 43 2-4-2 Reverse transcription 43 2-4-3 Polymerase chain reaction 44 2-5 RAT SCIATIC NERVE TRANSECTION AND TREATMENT 47 2-5-1 Gait analysis 48 2-5-2 Innervated muscle weight 49 2-5-3 DRG co-culture 49 2-6 HISTOLOGICAL STAINING 50 2-6-1 Hematoxylin and Eosin stain 50 2-6-2 Immunofluorescence stain 51 2-7 STATISTICAL ANALYSIS 51 CHAPTER 3. RESULTS 52 3-1 DEVELOPMENT OF DIFFERENT CELL SHEETS 52 3-1-Formation of Stable Cell Sheets Using Undifferentiated and Differentiated Cells 52 3-2MOLECULAR CHARACTERIZATION OF NERVE CELL SHEETS 56 3-2-1 qPCR revealed significant upregulation of neural markers NESTIN, GFAP, NFH, and S100β 56 3-2-2 Localization of GFAP and S100β validated the neural identity of the cell sheets. 58 3-2-3 Western blot analysis confirmed the presence of Nestin, GFAP and S100β, with GAPDH as internal controls. 61 3-3 FUNCTIONAL AND MOLECULAR VALIDATION OF MUSCLE CELL SHEETS 65 3-3-1 qPCR showed elevated expression of muscle differentiation-related genes, including Myod1, Myh4, Myh7, Myogenin, and ckm. 65 3-3-2 Localization of MHC and Rhodamine Phalloidin validated the muscular identity of the cell sheets 67 3-3-3 Western blot confirmed MyoHC and αSMA as primary markers of muscle cell sheets 69 3-4 EVALUATION OF NERVE REGENERATION OF CELL SHEET THERAPIES 72 3-4-1 Unaltered sciatic nerve transection models showed nerve disintegration and myelin loss. 72 3-4-2 Unaltered sciatic nerve transection models showed nerve disintegration and myelin loss. 74 3-5 EVALUATION OF CELL SHEET THERAPIES IN A RAT SCIATIC NERVE INJURY MODEL 76 3-5-1 Gait analysis revealed improved symmetry and motor coordination in cell sheet-treated animals. 76 3-5-2 Enhanced Expression of Regeneration Markers in Treated Nerve Tissues 80 3-6 H&E HISTOLOGY AND NFH/S100Β IMMUNOFLUORESCENCE REVEAL NERVE REGENERATION AND GLIAL ACTIVATION ACROSS NERVE SHEET TREATMENT GROUPS 84 3-7 H&E HISTOLOGY AND MHC/S100Β IMMUNOFLUORESCENCE REVEAL NERVE REGENERATION AND GLIAL ACTIVATION ACROSS MUSCLE-SHEET TREATMENT GROUPS 100 CHAPTER 4. DISCUSSION 109 CHAPTER 5. CONCLUSION 116 CHAPTER 6. REFERENCES 119

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