臺灣博碩士論文加值系統

摘 要

Sox1是早期表現在胚胎神經上皮細胞的一個轉錄因子之一，它的表現與早期胚胎發育過程中神經管的形成有關。此外也有研究指出Sox1會表現在胚胎telencephalon 及成體的lateral ventricle部位，且表達Sox1的神經前驅細胞也持續的增生中。因此，證明了Sox1的表現在早期胚胎發育過程與成體中的神經組織形成有關。
Sox1在老鼠胚胎中是神經前驅細胞最早期的一個專一性蛋白，在細胞完成有絲分裂後分化成神經元或神經膠狀細胞時表現量開始降低，為了探討不同培養時期的神經前驅細胞於活體內的分化潛能，因此需建立一個可活體追蹤的系統。利用病毒轉染的方式將DsRed紅螢光蛋白接在持續表現的EF1α(elongation factor 1α)啟動子後面，經過hygomycin篩選3~4週後可得到一個持續表達紅螢光蛋白的細胞株，將此細胞株在體外進行分化後利用流式細胞分選儀，我們可以分離出三個不同時間點表現sox1的神經前驅細胞。
將分離出之不同培養時期的神經前驅細胞移植到正常大鼠與中風大鼠的大腦皮質部位。在細胞移植一週後，我們可以觀察到在中風大鼠腦中，移植早期的神經前驅細胞有輕微向外遷移的痕跡，而在正常大鼠的腦中移植晚期(late-stage) 的神經前驅細胞幾乎都停留在移植部位。在細胞移植三週後，我們可以觀察到在中風大鼠腦中，移植早期的神經前驅細胞會沿著corpus callosum往損傷部位遷移。此外經由組織染色法我們可觀察到這些移植的細胞表現神經膠細胞專一性蛋白GFAP，表示移植的細胞分化為星狀神經膠細胞。綜上所述，我的結果顯示由胚胎幹細胞體外分化於不同時期的神經前驅細胞在正常及受損的腦中有不同的反應，經由上述這個系統提供了篩選由胚胎幹細胞分化而來的神經專一性細胞型態，可用來進一步探討幹細胞療法的可行性。

Abstract

Sox1 is one of the earliest transcription factors expressing in embryonic neuroepithelial cells and its expression correlates with the formation of the neural tube during early development. Additionally, it was demonstrated that Sox1 also expressing in the proliferating neural progenitors of both embryonic telencephalon and adult lateral ventricle at which active neurogenesis is evident. Taken together, these data indicate that Sox1 expressing cells may persistently contribute to neurogenesis from early development to adulthood.
Sox1 is the earliest specific marker of neural progenitor and it is down-regulated when the cells exit from mitosis and differentiate into neurons and glia. In order to investigate the survival and differentiation potential of neural progenitor cells derived from embryonic stem cells. We transfected a constitutive expressed red fluorescence protein under the control of human elongation factor 1α（EF1α） promoter into the 46C mES cells, which carry GFP knocked into Sox1 gene for in vivo tracking of the engrafted cells. After antibiotics screening, we obtained a stable constitutive expressed red fluorescence protein 46C mES cells. After further differentiation in vitro, we isolated Sox1 expressing neural progenitor cells form three different time point.
These isolated Sox1-expressing cells from different time point were then transplant into normal and ischemia lesioned cortex of rat. One week post-transplantation, we observed only the early stage NPCs migrated from transplantation site in lesioned cortex, whereas the late stage NPCs stay at the transplantation site in normal cortex. Three weeks post-transplantation, we observed engrafted early-stage NPCs migrated along the corpus callosum to the lesion site. Furthermore, these cells give rise to glia lineage which express glia marker, Glial fibrillary acidic protein. Taken together, our result showed that NPCs derived from different in vitro differentiation stage of ES cells processed different properties, in terms of , their in vivo response to brain injury, this finding together with the procedures created in the current research, provide an important first-step for selection of specific neural cell types from ES cells for stem cell based therapy.

目 錄

英文摘要…………………………………………………………………1
中文摘要…………………………………………………………………3
第一章 緒論
一、幹細胞……………………………………………………………5
1.幹細胞的特性與分類
2.胚胎幹細胞的分離及其分子特性
二、神經幹細胞………………………………………………………7
1. 神經幹細胞的來源
2. Sox1 knock-in 基因轉殖老鼠胚胎幹細胞
3. 胚胎幹細胞體外轉型為神經幹細胞
三、利用中風的動物模式探討神經前驅細胞移植的可行性………12
1. 缺血性中風的再生醫療
2. 神經幹細胞的趨化作用（Homing）與分子機制
四、研究動機…………………………………………………………16
第二章 實驗材料與方法
一、老鼠胚胎幹細胞的培養…………………………………………17
二、基因轉殖紅螢光蛋白……………………………………………20
三、類囊體(Embryo bodies)的形成…………………………………26
四、神經細胞的分化…………………………………………………27
五、利用流式細胞儀分離神經前驅細胞……………………………28
六、細胞移植…………………………………………………………29
七、腦組織切片………………………………………………………32
第三章 結果
一、建立可活體追蹤之報導基因……………………………………36
二、無血清的情況下誘導Sox1-eGFP的表現………………………42
三、類囊體細胞貼附生長情形及流式細胞儀分選效率……………46
四、中風誘導移植細胞的遷移………………………………………50
第四章 討論 ……………………………………………………………61
第五章 參考文獻 ………………………………………………………69

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