開發結合神經滋養因子梯度與許旺氏細胞的微米圖貌培養裝置應用於神經組織工程

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研究生：	葉佳偉 Yeh Chia Wei
論文名稱：	開發結合神經滋養因子梯度與許旺氏細胞的微米圖貌培養裝置應用於神經組織工程 Development of micro-patterned device incorporated with neurotrophic gradient and supportive Schwann cells for the applications in neural tissue engineering
指導教授：	王子威 Wang Tzu Wei
口試委員:	王潔 Wang Jane 蔡金吳 Tsai Jin Wu
學位類別：	碩士 Master
系所名稱：	工學院 - 材料科學工程學系 Materials Science and Engineering
論文出版年：	2015
畢業學年度：	104
語文別：	英文
論文頁數：	83
中文關鍵詞：	軸突引導、微米圖貌、神經滋養因子梯度、許旺氏細胞
外文關鍵詞：	Axonal guidance, micro-patterned surface, neurotrophic gradient, Schwann cells
相關次數：	點閱：2 下載：0
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周邊神經受損是由於周邊神經纖維以及其結締組織受損而造成周邊神經永久性功能喪失。近年來，人工神經導管被廣泛地開發作為周邊神經受損的替代療法，中空管狀型的結構可用來引導受損的神經沿著管壁從受損近端往遠端生長，進而重新連接受損神經，使其回復原本之功能性。然而，人工神經導管缺乏完整之細胞外基質以及支持性的神經膠細胞，因此神經再生修復的結果仍然不是相當完好。而新型的人工神經導管則開始加入不同的刺激因子，如物理性之方向性引導、化學性之神經滋養因子引導以及生物性之支持細胞引導等促進周邊神經之修復再生以及功能性回復。
本研究中利用生物可降解高分子， poly(glycerol-co-sebacic acid) (PGS)，作為培養裝置基材，並且結合三重刺激因子：1) 微米尺度的方向性圖貌作為物理性引導；2) 神經生長因子濃度梯度之明膠薄膜作為化學性引導；3) 同時培養重要神經膠細胞，許旺氏細胞作為生物性之內源性支持以及引導，探討已分化之神經幹細胞之軸突生長情形 (方向引導、軸突生長速度以及長度)，進而應用於新型神經導管之開發。結果顯示利用雷射剝蝕系統可以在PGS上剝蝕出具方向性之微米圖貌，並且神經細胞可以貼附在較大的圓形區域內，並且神經軸突有沿通道方向延伸的情形。許旺氏細胞則從成功地從大鼠坐骨神經中分離出來，藉由免疫螢光染色以及流式細胞儀分析，兩種許旺氏細胞的特異性蛋白，s-100β以及p75 low affinity receptor of nerve growth factor 均有大量的表現，證實許旺氏細胞可以在體外培養且具有原表現型，而與許旺氏細胞共培養之神經幹細胞亦會朝神經細胞分化成熟。此外，我們也成功製作出具有神經滋養因子濃度梯度分布之明膠薄膜，且神經軸突在神經滋養子高濃度區域的生長情形有明顯的提升。

The current gold standard treatment in clinic for peripheral nerve injury is autologous nerve graft. Although there is no immune response after graft and complete extracellular matrix and Schwann cells are preserved in the nerve tissue, limited amount of available nerve and possible neuroma formation accompanied with permanent functional loss at donor site are the main concerns. In these years, the nerve guidance conduit has been developed as an alternative way to repair peripheral nerve. However, without incorporating proper stimulating factors, the prognosis is still not very satisfied.
In this study, a biodegradable polymer (poly (glycerol-co-sebacic acid), PGS) based novel device has been developed and characterized. By incorporating three stimulating factors: 1) micro-patterned surface, that can directionally guide the axon as physical cue; 2) neurotrophic gradient membrane, that can continually attract axon outgrowth from the proximal to distal stump as chemical cue; 3) Schwann cells, that can support the growth of neurite and form myelin sheath around axon as biological cue, we expect that this scaffold can be used as a promising nerve guidance conduit for peripheral nerve regeneration. The results showed that the micro-patterned surface with specific dimensions of channels and chambers can be fabricated with good uniformity. Attachment and extension of differentiated neuron cells were observed in larger chamber area. The directional extension of neurite along direction of channel were also observed. By immunocytochemistry and flow cytometry, strong expression of s-100β and p75 low affinity receptor of nerve growth factor can be observed, indicating that Schwann cells can be successfully obtained from rat sciatic nerve and maintained their phenotype under in vitro culture environment. When co-culturing Schwann cells with neural stem cells, neural stem cells will differentiate toward neuronal cells. The gradient distribution of nerve growth factor 7S on gelatin membrane was successfully achieved. Significant enhancement of neurite area/field and length/field were also observed in higher concentration of NGF gelatin segment.

中文摘要    3
Abstract    5
Chapter 1. Introduction    15
1    Peripheral nerve injury and self-repair mechanism    15
2    Clinic treatment for PNI    18
3    Nerve guidance conduit    19
4    Stimulation factors    21
5    A biodegradable polymer: poly (glycerol-co-sebacic acid) (PGS)    24
6    Motivation and objective of this study    25
Chapter 2. Literature Reviews    27
1 The development of nerve guidance conduit    27
1.1 Non-biodegradable NGC    28
1.2 Biodegradable NGC    28
1.3 Biodegradable NGC with external stimulating factors    29
Chapter 3. Theoretical basis    36
1 Neurotrophic guidance and haptotaxis    36
2 Topographic guidance    38
Chapter 4. Materials and Methods    40
1 Materials (list)    40
2 Instrument (list)    41
3 Experimental design    41
4 Synthesis of poly (glycerol-co-sebacic acid) (PGS)    42
5 Ablation of micro-pattern on the PGS surface    42
6 Characterization of PGS    43
6.1 Inspection of micro-patterned PGS surface by scanning electron microscope    43
6.2 Surface property test of PGS    43
6.3 Degradation test of PGS    43
6.4 Tensile test of PGS    44
7 Oxygen plasma pre-treatment on PGS    44
8 Harvest of Schwann cells    45
9 Cell culture of neural stem cells (NSCs)    46
10 Characterization of Schwann cells and neural stem cells    46
10.1 Immunocytochemistry staining    46
10.2 Flow cytometry    47
11 Inspection of Schwann cells and neuron cells on micro-patterned PGS surface    48
12 Purification of microbial Transglutaminase (mTG)    49
13 Fabrication of concentration gradient gelatin membrane    50
14 Fabrication of NGF gradient on gelatin membrane    52
15 In vitro neurite outgrowth on NGF gradient gelatin membrane    53
16 Characterization of NGF release from gelatin membrane    53
17 Statistical Analysis    54
Chapter 5. Results    55
1 Characterization of PGS    55
1.1 Physichemical property of PGS    55
1.2 Degradation test of PGS    58
2 Micro-pattern ablation on the surface of PGS    59
2.1 Top view of mciro-patterned PGS surface    59
2.2 Cross-section view of micro-patterned PGS surface    60
4 Characterization of Schwann cells    62
4.1 Flow cytometry    62
4.2 Immunocytochemisty staining    63
5 Neural stem cell differentiate into mature neuron lineage    64
6 Schwann cells co-culture with neural stem cells    65
7 Concentration gradient of Rhodamine B on gelatin membrane    65
8 NGF gradient on gelatin membrane    67
9 Cumulative NGF release from gelatin membrane    68
10 Neurite outgrowth on NGF gradient gelatin membrane    69
11 Neurite outgrowth on micro-patterned PGS    70
Chapter 6. Discussion    72
1 Characterization of PGS    72
2 Schwann cell medium containing forskolin promote differentiation of neural stem cells into mature neuron    73
3 Schwann cell co-culture with neural stem cells    74
4 Cumulative release of NGF from gelatin membrane and enhanced neurite outgrowth on NGF gradient gelatin membrane    74
Chapter 7. Conclusion    77
Reference    78


                                

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