第一部分:利用層連結蛋白表面修飾之二氧化矽奈米纖維支架對神經幹細胞分化之影響
第二部分:探討沒食子兒茶素沒食子酸酯於脂多醣誘導帕金森氏症大鼠模型中神經保護作用

第一部分: 靜電紡纖維支架已廣泛應用於組織工程中。本論文的目的為觀察有無表面修飾層連結蛋白(laminin) 的順向與隨機二氧化矽奈米纖維支架對大鼠神經幹細胞（rNSCs) 神經分化的能力。研究中使用各種分析方法，如Trypan-Blue測試、MTS測定、QRT-PCR和免疫螢光染色，以評估支架對細胞貼附，細胞存活率和神經元特異性基因表達的影響。結果顯示在各組支架對神經幹細胞皆具有貼附能力。更重要的是，免疫螢光染色結果顯示其順向表面修飾層連結蛋白(Aligned2- laminin, A2/L)的二氧化矽奈米纖維支架大大增加了神經突長長度且大鼠神經幹細胞分化之突長會沿纖維的方向延伸。在神經元特異性基因表達分析中，A2/L支架上培養的大鼠神經幹細胞觀察到神經相關基因Tuj-1的表達量最高。並且發現與纖維排列方向無關，只要修飾層連結蛋白就可增強大鼠神經幹細胞分化為神經膠細胞的比例。根據上述實驗結果表示，經層連結蛋白表面修飾後的順向二氧化矽奈米纖維支架可做為神經組織工程優良候選材料。第二部分: 帕金森氏症（Parkinson’s disease, PD）是常見的神經退化性疾病，其特徵為腦部黑質區中多巴胺神經元的死亡所導致。越來越多的研究表明抑制微膠細胞發炎反應成為帕金森氏症治療與預防的研究方向。本實驗使用沒食子兒茶素沒食子酸酯((-)-epigallocatechin-3-gallate, EGCG) 是綠茶中主要的多酚成分，已知具有抗氧化、抗發炎以及神經新生作用的能力。在實驗中利用微脂體(Liposome)作為藥物載體，可延緩EGCG的釋放。本研究的目的是想了解Liposome-VE-EGCG在大鼠帕金森氏症模型中的神經保護作用。動物實驗中，我們利用脂多醣(lipopolysaccharide, LPS)注射至大鼠右腦黑質區，使誘導微膠細胞活化和多巴胺神經元的損傷，誘發成類帕金森氏症病理模型。並利用動物行為與生化分析Liposome-VE-EGCG是否夠減緩多巴胺神經元損傷與其神經保護之作用。在手術後四週的結果表示，Liposome-VE-EGCG能夠改善因安非他命所引發的旋轉行為。並與控制組相比其Liposome-VE-EGCG能夠顯著降低中腦黑質區神經發炎因子TNF-表達量。本研究結果顯示Liposome-VE-EGCG具有神經保護作用，可降低神經發炎因子表達與改善行為能力。因此EGCG可做為有效降低神經發炎反應的神經保護藥劑。

關鍵字

神經幹細胞；二氧化矽奈米纖維；神經分化；帕金森氏症動物模型；兒茶素；神經發炎

並列摘要

PartI: Electrospun fibrous scaffolds have been widely applied in tissue engineering. The objective of this study was developing aligned and random silica nanofiber scaffolds with and without laminin to evaluate the potential of rat neural stem cells (rNSCs) for neural differentiation. Herein, we used various methods such as trypan blue exclusion test, MTS assay, real-time polymerase chain reaction, and immunocytochemistry to evaluate the effects of the scaffolds on cell adhesion, cellular viability, and neuron-specific gene expression of the cells. The results show that the rNSCs cultivated on all groups of scaffolds were able to adhere. More importantly, fluorescence microscopy images illustrated that the scaffold with aligned 2-laminin (A2/L) fibers greatly increased the average neurite length and directed neurite extension of differentiated rNSCs along the fiber. Gene expression analysis demonstrated that the highest expression of neural-related genes, tuj1 was observed in rNSCs cultured on A2/L scaffolds. Other results indicated that the modification of laminin could enhance the glial differentiation of the rNSCs and it was independent of the fiber alignment. Based on the experimental results, the aligned nanofibrous silca scaffold with laminin could be used as a are superior candidates in neural tissue engineering. PartII: Parkinson’s disease (PD) is a common neurodegenerative disorder, which is characterized by the selective and progressive death of dopaminergic (DA) neurons in the substantia nigra. Increasing evidence suggests that inhibition of microglia-mediated neuroinflammation may become a reliable protective strategy for PD. (-)-Epigallocatechin-3-gallate (EGCG) is a major polyphenol in green tea, has been known to possess antioxidant, anticancer, and anti-inflammatory properties. We Used liposome as a drug carrier, which can prolonged release of the EGCG. The aim of this study was to investigate the neuroprotective effect of liposome-VE-EGCG in a rat model of PD. Microglial activation and the injury of dopaminergic neurons were induced by LPS intranigral injection. Animal behavioral tests and biochemical assays were performed to evaluate the dopamine neuron degeneration and neuroprotective effects of liposome-VE-EGCG. Liposome-VE-EGCG significantly reduced amphetamine-induced rotational behavior in LPS-lesioned rats after 4 weeks. Furthermore, Liposome-VE-EGCG significantly decreased TNF-α levels, a marker of neuroinflammation in PD rats compared with saline group. These findings suggest that liposome-VE-EGCG exerts neuroprotection against LPS-induced dopaminergic neurodegeneration, and TNF-α.Thus EGCG represents a potent and useful neuroprotective agent for inflammation-mediated neurological disorders.

並列關鍵字

Neural stem cells ； silica nanofiber ； neural differentiation ； (-)-Epigallocatechin-3-gallate (EGCG) ； neuroinflammation

參考文獻

Biber, K., Neumann, H., Inoue, K., and Boddeke, H.W. (2007). Neuronal ‘On’and ‘Off’signals control microglia. Trends in neurosciences 30, 596-602.

Chen, W.S., Hsieh, P.H., Yang, W.N., Fan-Jen, P.Z., Yang, M.-L., Yeh, J.M., Wei, Y., Chin, T.Y., and Chen-Yang, Y.W. (2014). Chemically modified electrospun silica nanofibers for promoting growth and differentiation of neural stem cells. Journal of Materials Chemistry B 2, 1205-1215.

Chen, Z. (2015). Cell Therapy for Parkinson’s Disease: New Hope from Reprogramming Technologies. Aging and disease 6, 499.

Christopherson, G.T., Song, H., and Mao, H.-Q. (2009). The influence of fiber diameter of electrospun substrates on neural stem cell differentiation and proliferation. Biomaterials 30, 556-564.

de Oliveira, M.R., Nabavi, S.F., Daglia, M., Rastrelli, L., and Nabavi, S.M. (2016). Epigallocatechin gallate and mitochondria—A story of life and death. Pharmacological research 104, 70-85.

國際替代計量

第一部分:利用層連結蛋白表面修飾之二氧化矽奈米纖維支架對神經幹細胞分化之影響第二部分:探討沒食子兒茶素沒食子酸酯於脂多醣誘導帕金森氏症大鼠模型中神經保護作用

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