生醫材料與培養基成份對神經幹細胞/前驅細胞行為之探討

Abstract

神經幹/前驅細胞可以在懸浮於基材表面上的情況下增生並形成神經幹/前驅細胞球，它被認為是一種非下錨型的細胞。然而，神經幹/前驅細胞球則被認為是在適當條件下貼附於基材上時，會進而分化成不同種類細胞的一種下錨型細胞。本研究的目的是藉由結合培養基和生醫材料來調控神經幹/前驅細胞的增生與分化。眾所皆知血清中具有許多促進增生或分化的分子可以對神經幹/前驅細胞的行為產生極大的影響。因此本論文中分為兩的主要的部份。 第一部份為探討血清的角色，10%的胎牛血清及其部份成份被加至培養基之去評估對於胚胎鼠大腦皮質所分離的神經幹/前驅細胞的分化潛能之影響。當神經幹/前驅細胞被分化到七天之後，利用免疫螢光染色去標定神經系細胞的特定蛋白，可以發現到當培養基中含有血清中小於分子量100 kDa的分子和存在纖維母細胞生長因子時可以促進神經幹/前驅細胞往神經元的分化。相反的，神經幹/前驅細胞則是在存在全血清成份時，其分化的細胞主要為膠質細胞。由半定量來分析免疫染色的結果，當神經幹/前驅細胞存在於結合血清成份小於100 kDa、聚乙烯乙烯醇共聚物 (EVAL)和纖維連接蛋白可以發現爬出球外的細胞具有神經元表現的比例被促進到約有85%之多，此外藉由中腦中央動脈阻塞的動物中風實驗模型， 也可以發現到分清的成份，分子量小100 kDa 可以促進神經元的生成及中風動物的行為功能回復。 論文中的第二部份是10%的胎牛血清及其成份被加至培養基之去評估對於胚胎鼠大腦皮質所分離的神經幹/前驅細胞的貼附及增生潛能之影響。文中顯示上皮生長因子和纖維連接蛋白對神經幹/前驅細胞具有一個協同效應。而當血清成份存在於培養基中時，結合上皮生長因子和纖維連接蛋白比其單一成份對神經幹/前驅細胞的影響有明顯的差別。有趣地當血清中存在上皮生長因子和纖維連接蛋白時，神經幹/前驅細胞會快速地貼附於細胞培養盤上而且爬出來的細胞有高於70%維持著巢蛋白的表現。而這些在具有貼附形態的未分化巢蛋白表現的神經幹/前驅細胞仍可以被誘導分化成其他神經系的細胞。這些結果提供了結合了上皮生長因子和纖維連接蛋白可以有效地將神經幹/前驅細胞在貼附的情況下維持巢蛋白的表現，和可以做為一個線索去研究血清成份對神經幹/前驅細胞不分化的反應。更進一步地，結合上皮生長因子、纖維連接蛋白和高分子導管，也可以讓神經幹/前驅細胞在導管中維持不分化的狀態，這樣顯示可能可以被使用在神經導管的應用上。這些結果是令人鼓舞的， 因為當想要一個神經元分化或是維持神經幹細胞不分化的環境時，我們可以在神經科學研究的領域中提供有用的策略去控制神經發育的過程。

Neural stem/precursor cells (NSPCs) can proliferate in suspension to form neurospheres that do not need to attach to the substrate surface and, thus, can be considered as anchorage-independent cells. However, neurospheres are considered to be anchorage-dependent cells, which differentiate into different cell types when they attach to the substrate under appropriate conditions. The purpose of this study was to regulate the proliferation and differentiation of NSPCs by the combination of media, biomaterials. It is known that many proliferation- and differentiation-promoting molecules are present in the serum, which has a great effect on the behaviors of NSPCs. There are two major parts in this thesis. One is that considering the role of serum, 10% fetal bovine serum or its fractions were added to DMEM/F12 medium to examine the effect of the differentiation-promoting potential on cultured NSPC isolated from embryonic rat cerebral cortex. The NSPCs were cultured for 7 days, after which differentiation was assayed using immunocytochemistry for neural lineage specific markers. It was demonstrated that molecules promoting neuron differentiation were present in serum with molecular weight <100 kDa, which could dominate the differentiation of NSPCs principally into neurons in the presence of basic fibroblast growth factor (bFGF). In contrast, NSPCs were induced to differentiate predominantly into glial cell phenotypes in the presence of whole serum components. Based on medium containing serum fraction, semi-quantification showed that the MAP2-positive percentage of the immunoreactive ratio within migrated cells could be promoted over 85% by combining poly(ethylene-co-vinyl alcohol) (EVAL) biomaterial and fibronectin matrix protein. In addition, by the middle central artery occlusion (MCAO) rat, It also be demonstrated that the component of serum with molecular weight <100 kDa could promote the generation of neuron in the ischemia brain and recover the function of MCAO rat. The other part in the thesis is that 10% FBS or its components were added into culture medium to examine the effect of the adhesion- and proliferation-promoting potential on NSPCs. It appeared that medium containing epidermal growth factor (EGF) and fibronectin had a synergistic effect to regulate NSPCs, and cooperation of EGF and fibronectin was more significant than singly effect in regulation of NSPCs when serum components were present in the culture system. Interestingly, when culture medium was in the presence of EGF and fibronectin, NSPCs rapidly attached onto the TCPS surface and more than 70% migrating-out cells were predominantly maintaining nestin-positive phenotype. Furthermore, the result of undifferentiated nestin-positive dominating the pattern of NSPCs adhesion could be induced to differentiate into neural linage cells. These results informed that the combined administration of serum components with EGF and fibronectin could significantly maintain nestin-positive of adhesive NSPCs phenotype, and will therefore be an important clue to investigate what molecules in serum are responsible for the NSPCs undifferentiation-maintaining activity. Furthermore, combination of the medium containing EGF and fibronectin and polymer conduit also could maintain NSPCs at undifferentiated phenotype in the conduit, which might be applied to be a neuronal conduit. These results are very encouraging, since an environment favorable for neuronal differentiation and maintenance of undifferentiated NCPSs should be useful in the development of strategies for controlling the behavior of NSPCs in neuroscience research.