Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52865
標題: 維持Sox1+神經母細胞之全神經分化能力
Maintenance of the Pluripotency of Sox1+-Neuroblasts
作者: 蘇鴻麟
關鍵字: 生物技術;基礎研究
摘要: 
動物胚胎在原腸期(gastrula) 分化為神經細胞時, 首先藉由抑制BMP(bonemorphogenetic protein) 的路徑誘導外胚層細胞分化為初始神經母細胞(primitiveneuroblasts)。此時的神經母細胞只會分化為神經細胞(neurons),與腦室附近的神經幹細胞(neural stem cells, NSC)。早期分化的神經包括有中腦的多巴胺神經細胞與中後腦交界的serotoninergic 神經元。在神經形成的後期,此時不表現sox1基因的神經幹細胞可再分化為大腦皮質外層的神經細胞與神經膠細胞(glia cells)。NSC的體外培養,可受EGF(epithelial growth factor)與FGF2(fibroblast growth factor 2)的刺激,而自我複製與增殖。相對於初始神經母細胞,NSC的子代則多為神經膠細胞與少部分的神經細胞,與晚期胚胎中的radial glia cells相似。雖然目前可以利用EGF與FGF2來維持NSC的神經分化能力,但如何維持早期sox1+神經母細胞的不分化則少有研究。本計畫將利用小鼠來源之sox1-GFP Knock-in胚幹細胞及建立人類sox1-GFP knock-in胚幹細胞,於體外誘導出最早期之神經母細胞, 及建立神經發生誘導的環境。利用cell sorting 與antibiotics-selection的方法純化Sox1-GFP+ cells,以及研究leukocyte inhibitory factor、Wnt3a、sonic hedgehog、anti-bone morphogenetic protein及FGF2等分子,對於維持Sox1+神經母細胞的多能性(multipotency)的影響。本計畫也將探討以上因子在小鼠與人類胚幹細胞誘導出的神經母細胞的差異。此外,目前已知一染色體licensing factor,geminin,可能與Brg1作用,共同維持神經母細胞的不分化與自我複製。藉由四環黴素誘導系統來控制geminin與Brg1兩個基因,於胚幹細胞由來之Sox1+-GFP神經母細胞中表現,觀測sox1 promoter所驅動的GFP的表現,可以進一步探討相關訊息傳遞與分子機轉。研究初始原始母細胞多能性的維持不只對於神經的早期發生的基礎研究甚為重要,臨床應用上,也可利用patterning factor的刺激,使初始神經幹細胞分化為中腦的多巴胺神經元。本研究也將加速日後由ES細胞產生特定神經細胞的研究及應用於細胞移植的治療。

During vertebrate gastrulation anti-bone morphogenetic protein (BMP) signals inducethe neurogenesis of uncommitted ectoderm into neuro-ectoderm. Primitiveneuroblasts, expressing the sox1 transcriptional factor, differentiate into neurons andneural stem cells (NSCs) near the ventricular zone. These early-stage maturedneurons include the dopaminergic neurons in the midbrain and serotoninergicneurons in the midbrain-hindbrain boundary, etc. In late-stage of neurogenesis,Sox1 negative NSCs differentiate into out-layers neurons of the cortex and glia cells.In vitro clonal expansion of the NSCs and neurosphere formation are established bythe helps of epidermal growth factor (EGF) and fibroblast growth factor (FGF) -2mediated growth stimulation. Correlated to the radical glia cells in vivo, most of theprogenies of NSCs are glia cells, rather than neurons. Although EGF and FGF2signals are essential for the self-renewal of NSCs, it is still obscure for themaintenance of multipotency of Sox1+ primitive neuroblasts. In this project,Sox1-GFP knock-in mouse embryonic stem (ES) cells and Sox1-GFP knock-in humanES cells will be generated and subjected for the generation of Sox1+ neuroblasts byusing the efficient neural induction procedure and consequent FACS-sortingenrichment and antibiotics selection. Several candidate factors will be tested for theconstant expression of sox1 gene in neuroblast cells, including leukocyte inhibitoryfactor (LIF), Wnt3a, sonic hedgehog, anti-BMP factors and FGF2. The variations ofthese candidate factors between mouse and human Sox1-ES cells will be carefullyevaluated. It has been demonstrated that geminin, a chromosome licensing factor,sequesters Brg1 to maintain the multipotency of primitive neuroblasts in Xenopusembryo. A tet-on inducible system for the geminin and Brg1 expression will beestablished in Sox1 GFP knock-in mES cells and hES cells and further evaluate thesox1-driven gene expression and related proneural genes profile. The study ofmaintenance of pluripotency of sox1+ neuroblast is important for the basic braindevelopment and further clinical application, such as efficient generation of abundantdopaminergic neurons. This study will also accelerate the research for thespecification of neural fates and cell-transplantation based therapy.
URI: http://hdl.handle.net/11455/52865
其他識別: NSC96-2321-B005-007-MY3
Appears in Collections:生命科學系所

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