幾丁聚醣於神經修復與再生之應用:細胞來源與材料篩選

Abstract

神經系統疾病或損傷可能會導致部分運動或感覺功能喪失。目前臨床的治療方式有很多種，例如幹細胞治療、神經導管移植等。而不同部位的神經損傷，其治療模式也不盡相同。雖然上述的治療模式都已被應用於神經修復上，然而每種治療方法都有其限制。因此本論文著重探討不同細胞來源與幾丁聚醣材料對中樞及周邊神經的修復，論文將分為四個部分。第一部分探討由幾丁聚醣基材培養所獲得之幹細胞球體對氧化鐵奈米粒子攝入的影響，並利用此技術建立體內細胞追蹤之平台。研究中發現幹細胞培養於幾丁聚醣基材上能增加氧化鐵奈米粒子之攝入量，細胞對奈米粒子之耐受性也較一般培養基材(TCPS)高。推測原因為幹細胞於幾丁聚醣基材上可促進自噬反應進而避免幹細胞在環境壓力下走向死亡。接著將氧化鐵標定之幹細胞植入斑馬魚體內，相較於TCPS培養之幹細胞，幾丁聚醣所獲得之幹細胞球體有較高的存活率且可長時間追蹤。此結果顯示出幹細胞培養於幾丁聚醣基材上能使奈米粒子有效率的被攝入，且所獲得之幹細胞球體於動物實驗應用上帶來高移植率並可長時間追蹤之優勢。第二部分為探討幾丁聚醣所獲得之間葉幹細胞球對周邊神經修復的影響。基於第一部分之結果，為追蹤間葉幹細胞於神經修復上所扮演的角色，我們將氧化鐵奈米粒子標定之間葉幹細胞球與神經導管共同植入到大鼠受損的坐骨神經，利用核磁共振造影(MRI)於體外追蹤移植的細胞，觀察神經再生的情形。結果發現神經幹細胞球(藉由幾丁聚醣基材所得)可有效修復受損的周邊神經；氧化鐵標定之間葉幹細胞球體可以被MRI所追蹤，可觀察周邊神經再生的過程。此研究顯示幹細胞球體對周邊神經的修復較一般分散的幹細胞好，故利用幾丁聚醣基材轉染質體或是傳遞奈米粒子至間葉幹細胞可為細胞治療帶來新的附加價值。第三部分則進一步的利用幹細胞球體應用於中樞神經損傷之治療。在此部分，我們將自修復且可注射的幾丁聚醣水膠包覆神經幹細胞球體，發現幹細胞球體於水膠內有良好的生長與神經分化的能力。接著將其注射至中樞神經受損之斑馬魚體內，結果發現此一方式，不僅可以增加細胞於生物體內之植入率，更可以提升修復受損的中樞神經。第四部份則是解決神經細胞來源不足的問題。利用幾丁聚醣基材將FOXD3質體轉染至纖維母細胞，使細胞重新編程為類神經脊幹細胞，並應用於中樞神經損傷之修復實驗。結果發現轉染FOXD3之纖維母細胞其神經脊相關表現、幹性、神經相關表現都較未轉染的細胞高。進一步將轉染FOXD3的細胞移植至中樞神經受損之斑馬魚，發現轉染FOXD3的細胞能增加受損神經回復的能力。此研究顯示出利用幾丁聚醣基材可將體細胞重新編程，取代神經相關細胞來源不足之困境，進而應用在治療神經疾病。透過本論文四個部分探討細胞來源與材料之選擇對於周邊與中樞神經修復的影響。利用幾丁聚醣傳遞基因與奈米粒子至細胞或用幾丁聚醣水膠包覆細胞是安全且有效的方式，且證實幾丁聚醣在神經修復極有應用性。

Neurological disorders or nerve injuries may result in a partial or complete loss of motor and sensory functions. Current clinical treatments for nerve repair include stem cell-based therapy, artificial nerve guides, and etc. Different types of nerve injury will determine the type of treatments. Although various approaches have been used to repair nerve injuries, there remain some drawbacks. Herein, this study tried to investigate different cell sources and biomaterial selection in nerve repair. In the first section, the mechanism for having the higher cellular uptake as well as better cell survival on the chitosan substrates was studied. We found that cells cultured on the chitosan substrate may be more tolerant to NP cytotoxicity by the increased autophagy response. In animal studies, cells grown on chitosan had better survival after transplantation than those grown on TCPS. The increased survival of labeled cells may facilitate long-term cell tracking. This part of study suggested that chitosan as a culture substrate can induce cell autophagy to increase cell survival in particular for NP-labeled cells. This will be valuable for the biomedical application of NPs in cell therapy. In the second section, the effect of the substrate-derived MSC spheroids vs. single cells on the regeneration of transected rat sciatic nerve was evaluated. Results showed that MSC spheroids were superior to single cells in regeneration of transected peripheral nerve, especially for the BDNF-transfected MSC spheroids. Besides, Fe3O4 NPs-labeled MSC spheroids in the conduits were successfully tracked by MRI. The above findings indicated that the substrate-mediated Fe3O4 NP labeling for MSCs may be generally used as a bioimaging tool in animal studies. The substrate-mediated gene/NP delivery may equip MSC spheroids with extra values in carrying the therapeutic/diagnostic agents for cell-based therapy. In the third section, we sought to determine if cells combined with a chitosan-based self-healing hydrogel may offer therapeutic potentials for treating neurological disorders. Results showed that NSC spheroids grew twice faster in self-healing hydrogel compared to conventional alginate gel and had a greater tendency to differentiate into neuron-like cells. In the zebrafish embryo neural injury model, injection of the chitosan-based self-healing hydrogel with NSC spheroids produced a remarkable healing effect on neural development. These promising data suggest the potential of the novel injectable, biodegradable, self-healing hydrogel in repairing the central nervous system. In the last section, we sought to determine if FOXD3 delivered by the substrate-mediated method could reprogram human fibroblasts into neural crest-associated cells for potential treatment of neurological disorders. Results showed that cells could be reprogrammed into multipotent neural crest stem-like cells with self-renewal and differentiation capacity by a simple, safe, chitosan substrate-mediated FOXD3 transfection. The reprogrammed cells demonstrated functional rescue for impaired CNS in zebrafish models. The reprogrammed fibroblasts with neural crest stem-like behavior may be used as an easily accessible cellular source for treating neural diseases in the future. Through the above findings, cell spheroids combined with chitosan substrate-mediated NP/gene delivery, or encapsulated in chitosan hydrogel had a great therapeutic potential in nerve repair.