大鼠胚胎之大腦皮層神經幹細胞在高分子基材上行為之探討

The behavior of embryonic rat cerebral cortical neural stem cells on the polymer substrates

洪智煌(Chih-Huang Hung)

指導教授 ： 楊台鴻

國立臺灣大學/工學院/醫學工程學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.02999

摘要

本研究探討大鼠胚胎之大腦皮層神經幹細胞在高分子聚乙烯乙烯醇、聚乙烯醇以及離氨酸-丙氨酸序列共聚物基材上行為之調控。在體外培養時，神經幹細胞的行為受到下列三種因素所調控: (1) 培養基中的可溶性因子，(2) 細胞與細胞之間的交互影響，(3) 培養的基材，因此，藉由改變這三者之間的交互作用來控制神經幹細胞在生醫基材上的行為與生長。
第一章介紹幹細胞、中樞神經系統、中樞神經系統之神經幹細胞、影響神經幹細胞生長行為的因素、神經幹細胞的免疫化學分析、生醫基材、以及神經幹細胞的應用。
第二章探討神經幹細胞在沒有血清的條件下，其在高分子聚乙烯乙烯醇以及聚乙烯醇薄膜基材上行為之調控。在纖維母細胞生長因子的作用下，神經幹細胞會因細胞與細胞之間交互作用的強度，而改變生長方式。當以單顆神經幹細胞培養在聚乙烯乙烯醇基材上時，也就是細胞與細胞之間的交互作用較小時，細胞會貼附於基材上，但不分化，根據染色結果顯示，仍保有高比例的神經幹細胞。另外，當以低密度之神經幹細胞團培養時，神經幹細胞團會貼附至聚乙烯乙烯醇基材上並被誘導分化；假如繼續提高神經幹細胞的密度時，也就是提高細胞與細胞之間的引力，此時神經幹細胞並不會貼附於基材上，反而懸浮於基材上並被誘導增生。另外，結果也顯示在此條件下，神經幹細胞無法存活於聚乙烯醇薄膜基材上。
第三章則探討神經幹細胞團在以丙氨酸-離氨酸序列共聚物為基材上行為之調控，結果顯示在沒有血清的條件下，神經幹細胞團會因為培養時密度的不同，而採用不同的方式與鄰近的細胞聯繫。另外，當血清加入到此系統中後，因為血清對細胞的效應大過於基材對細胞的效應或細胞與細胞之間的效應，此時神經幹細胞已呈現不同的生長方式。
第四章則是探討當血清直接加至培養基中或以塗佈的方式塗佈於聚乙烯乙烯醇與聚乙烯醇薄膜基材對神經幹細胞行為的影響。結果顯示培養基中的血清與塗佈的血清對於神經幹細胞的分化各有不同的影響，然而此時所使用的基材會影響神經幹細胞分化的細胞類型。
第五章則是探討神經生長因子對於培養在聚乙烯乙烯醇基材上之神經幹細胞團的影響，結果顯示，只有在沒有血清條件下，且在神經生長因子的作用下，神經束才會形成束狀生長。
第六章總結本研究的貢獻，藉由調控神經幹細胞生長的微環境，以此控制神經幹細胞在生醫材料上的行為包括誘導增生、分化。

關鍵字

神經幹細胞 ； 聚乙烯乙烯醇 ； 聚乙烯醇 ； 離氨酸-丙氨酸序列共聚物 ； 胎牛血清 ； 神經生長因子

並列摘要

In this study, the behavior of embryonic rat cortical neural stem cells on biomedical polymer substrates, poly-(ethylene-co-vinyl alcohol) (EVAL), polyvinyl alcohol (PVA), and lysine-alanine sequential (LAS) were explored. However, the behavior of neural stem cells in vitro are mediated by three major factors: (1) soluble factors in the medium, (2) cell-cell interactions, and (3) culture substrates. Hence, by means of altering the interactions of these three factors, the behavior of cortical neural stem cells was manipulated on these substrates.
Chapter one introduces the backgrounds of stem cells, central nervous system (CNS), neural stem cells from CNS, the factors regulated the behavior of neural stem cells, immunocytochemistry for neural stem cells, biomedical polymer substrates, and application of neural stem cells.
Chapter two investigates the behavior of neural stem cells on PVA and EVAL membranes in the presence of the mitogenic effect of basic fibroblast growth factor (bFGF) in the serum-free medium. It was found that EVAL and PVA membranes exerted different influences on the fate of neural stem cells. The behavior of neural stem cells on the EVAL membrane was independent of cell density at the single-cell level. Conversely, the development of cell clusters was in a density-dependent manner on the EVAL membrane. Neurospheres continuously proliferated under high-density culture condition, but differentiated into neurons and astrocytes under low-density culture condition. Therefore, it is reasonable to assume that biomaterials may stimulate or inhibit the proliferation and differentiation of neural stem cells.
Chapter three explores the phenotypic potential of cortical neural stem cells by inducing differentiation on LAS polymer substrates at neurosphere-level. The results provided evidences that LAS could improve better process growth of neurospheres than poly-D-lysine could. The results also suggested that the behavior, proliferation, and differentiation of neural stem cells on LAS substrates is responsible for the effects of seeding density and serum proteins. It was found that forming-neurospheres cells cultured on LAS substrates adopted different strategies to communicate with adjacent neurospheres
Chapter four investigates the influence of fetal bovine serum (FBS) adsorbed to EVAL and PVA substrates (coated FBS) and fetal bovine serum present in the culture medium (soluble FBS) on the behavior of cortical neural stem cells at neurosphere level. The results of this study suggest that besides substrates, coated and soluble serum proteins have their unique effect on the morphological differentiation and fate determination of neural stem cells.
Chapter five explores the effects of cell-cell and cell-substrate interactions on developmental potential of neural stem cells at neurosphere level in the presence of nerve growth factors (NGF) on tissue culture polystyrene (TCPS), poly-D-lysine (PDL), and EVAL substrates. The results also suggested that, in combination with NGF, EVAL substrates could induce process fasciculation of differentiated forming-neurosphere cells under serum free conditions. The results also provided evidence that EVAL can be a potent controller of cell interactions involving cell-cell and cell-substrate contacts. We proposed that, in combination with nerve growth factors, EVAL substrates could induce neurite fasciculation of embryonic rat cortical neural stem cells under serum free conditions.
Chapter six concludes the contribution of this study and suggested that the present study provides evidence that the plasticity of multipotential neural stem cells is versatile and is dependent on the complex environmental conditions.

並列關鍵字

neural stem cells ； poly-(ethylene-co-vinyl alcohol) ； polyvinyl alcohol ； lysine-alanine sequential ； fetal bovine serum ； nerve growth factor

參考文獻

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