應用幹細胞、神經生長因子與神經導管在神經修復之研究與應用

Abstract

神經損傷會造成神經細胞的死亡及神經纖維的斷裂，導致神經功能的喪失，且神經系統的高度分工性與複雜的損傷修復機轉，都造成神經再生過程的挑戰。本研究的目的，在運用組織工程的概念，配合神經損傷的修復策略，以生物分解性材料幾丁聚醣，來整合細胞、支架與細胞調控因子的三大組織工程元素達到神經再生的目的。首先應用幾丁聚醣與肝素，製備藥物釋放載體，來吸附、保護、延長並強化生長因子的生物效用，以神經滋養因子aFGF進行測試，載體可有效保護aFGF並達到長期釋放的目的，且對aFGF的吸附率高達85%以上。在動物實驗中，更發現載體本身還具有減少損傷組織纖維化的能力。另外透過複合材料與表面改質技術，將奈米碳管與幾丁聚醣結合，結果顯示，奈米碳管的高機械強度與導電性，可改善幾丁聚醣所製備的神經導管強度不足的問題並賦予導管導電的特性。由此ㄧ性質，期望未來能配合電刺激，用以促進神經再生的治療上，而細胞相容性不足的問題，則運用氧氣電漿改質的技術，來增加材料表面的親水性，更可以共價性鍵結的形式，與細胞外基質(ECM)等分子，在原本材料表面形成穩固的修飾，來創造更適合細胞生長的微環境，經PC-12細胞的測試，顯示確實可達到增加細胞貼附與神經分化的效果。本研究最後的實驗，則是運用骨髓幹細胞合併經昆布氨酸（laminin）修飾的幾丁聚醣導管，進行動物實驗。結果顯示，合併幹細胞療法，可明顯促進神經再生跨越1公分長的坐骨神經缺損，其運動神經元存活的數目亦較多。但是結果也發現幾丁聚醣在體內降解過程，可能會引發慢性的發炎反應，導致神經再生失敗，而合併幹細胞治療，則可調控發炎反應，達到再生的目的。而此動物實驗的結果，更凸顯生物實驗的重要，唯有透過長時間的生物試驗才能更確認研究成果的安全性與應用性，此乃材料或細胞層次的測試無法達到的。本論文運用各種神經策略進行神經再生的研究，循序漸進由材料、生長因子的分子層次到細胞，乃至於動物實驗，希望本研究之成果，能對神經再生的研究有些微的貢獻。

Nerve injury causes the death of neural cells and the breakage of nerve fibers resulting in the loss of nerve function. Neuron regeneration faces a difficult challenge due to the highly specialized system and the complex repair mechanism. In this study, according to the concept of tissue engineering and neural regeneration strategy, We use chitosan as a degradable biomaterial to integrate the three main tissue engineering elements: cell, scaffold and cell regulating factors, for achieving the purpose of nerve regeneration. Firstly, self-assembled drug carrier by using chitosan and heparin to adsorb, protect, prolong and enhance the bioactivity of growth factors (also called acidic fibroblast growth factor (aFGF)) was developed and also decrease the fibrosis and prevent adhesion in vivo. Furthermore, chitosan incorporating with carbon nanotube (CNT) can effectively improve the physicochemical properties of chitosan in various applications, especially in mechanical strength and electrical conductivity. In order to make CNT/Chitosan cell friendly most, electric O2-plasma treatment and laminin modification were applied. Successful modification was confirmed by immunolocalization, significantly improved cell adhesion and neurite extension. We hypothesize that CNT/chitosan materials provide functional nerve conduit for healing injured nerves. In this study, the final test used laminin-modified chitosan multi-walled nerve conduit combining with bone marrow stem cells (BMSCs), and grating to bridge in sciatic nerve of SD for 16 weeks. The result is shown that the therapy with stem cell can promote the neuron regeneration to crossover a 10 mm long gap and help more motor neuron to survivor. Moreover, the result is also shown that the degradation of chitosan might cause chronic inflammation which might fail the regeneration of neuron, and the therapy with stem cell can modulate the inflammation to overcome this problem. At last, this study is shown the important of animal experiments: because these data cannot present the material or cell test, the long-term animal experiments is the only way to confirm the safety and the applicability of research results. This paper using many strategy of neuron regeneration from materials, growth factors, cells to animal testing. Hope the results in this study can contribute a little to the research of neuron regeneration.