臺灣博碩士論文加值系統

脊椎動物的早期神經發育是經由囊胚期胚胎(blastocyst)中ICM(inner cell mass)衍生之epiblast而來，因為epiblast可以分化成外胚層(ectoderm)、中胚層(mesoderm)和內胚層(endoderm)三種不同胚層的細胞，其中神經系統是由外胚層發育而來。外胚層會先形成neuroectoderm進一步發育成neural plate，最後成為中樞神經系統的神經元來源。在以往的研究顯示Pax6 、Neurogenin、Sox1、Nestin等基因在老鼠早期neuroectoderm形成上扮演了重要角色，其中Sox1 最早表現於neuroectoderm，並能調節神經細胞的生成，但Sox1在人類發育中的角色，目前為止並不了解。所以我希望利用人類的胚胎幹細胞探討Sox1 基因在發育過程中的角色，我利用Sox1 RNA干擾(RNA interference) ，抑制Sox1 基因的表現，以探討其功能。另外，在脊椎動物早期神經分化系統中的前驅細胞(progenitor cells)時期的細胞族群中會有細胞異質性(cellular heterogeneity)的存在，先前文獻中顯示細胞異質性可能是造成發育中細胞多樣性的主要原因。所以我們利用Real-time PCR來探討神經分化過程中Oct4、Pax6 和Sox1基因在單一細胞(single cell)的變化。在我的實驗結果中發現所選擇的Sox1 siRNA片段不影響胚胎幹細胞的特性和神經細胞的分化。而Real-time PCR分析單一顆細胞的結果中顯示在早期神經分化過程中每顆細胞所表現的Oct4 , Pax6 和 Sox1基因模式並不相同，這樣的結果說明在早期發育時期中具有神經細胞的多樣性的存在。

The generation of functional neuronal subtypes in the vertebrate central nervous system involves several steps. Early vertebrate neural development started from epiblast which is the pluripotent descendents of inner cell mass(ICM) of the blastocyst. The epiblast cells give rise to three embryonic germ layers(ectoderm,mesoderm and endoderm). Subsequently, ectodermal precursors forms early neuroepithelium containing very early neural stem cells. Then, neuroectoderm forms the neural plate and neural tube which become the source of the central nervous system. Recently, it has been demonstrated that Pax6 , Neurogenin, Sox1 and Nestin genes play an important role in early development of murine neuroectoderm. Among these genes, Sox1 is the earliest gene to be expressed in neuroectoderm and plays essential role in neural specification. However, the function of Sox1 gene in the development of human is still unknown. In order to understand the functional role of Sox1 in human neural specification, I generated lenti-viral Sox1 RNAi clones with GFP reporter and transducted them into hESCs to knowdown the expression of Sox1. In addition, heterogeneity among progenitor cells in the vertebrate nervous system has been demonstrated and it is believed that celluar heterogeneity is an important step toward developmental diversity. Therefore, I also investigated the expression profile of Oct4,Pax6 and Sox1 gene of single hESCs and their differentiated derivatives. My results show Sox1 siRNA knockdown has no obvious effect on altering characteristics of hESCs and their potentials in neural differentiation. Furthermore, results of the single cell analysis indicated the expression pattern of Oct4,Pax6 and Sox1 genes are not uniform in individual cells of early neural differentiation. This may imply a diversified neural cell types are emerging at these developmental stages.

中文摘要....................................................................................................1
英文摘要....................................................................................................3
第一章 緒論
一、 幹細胞(Stem cell)
1. 幹細胞特性....................................................................................5
2. 幹細胞的分類................................................................................6
3. 胚胎幹細胞的發展與特性............................................................8
二、 神經發育與神經幹細胞(Neural development and Neural
stem cells)
1. 神經發育(Neural development) ..................................................11
2. 神經幹細胞(Neural stem cells) ...................................................12
3. 早期神經細胞的基因表現..........................................................16
三、 核醣核酸干擾(RNA interference；RNAi) ...............................17
四、 幹細胞的異質性(Heterogeneity) ..............................................19
五、 研究動機....................................................................................20

第二章 材料與方法
一、材料
1. 試劑與溶液...................................................................................22
2. 免疫細胞螢光(Immunofluorescence)染色之抗體.......................26
3. 引子(primer) .................................................................................27
4. 細胞株...........................................................................................28
5. 培養液...........................................................................................28
二、方法
1. 細菌的培養...................................................................................30
2. 甘油保存液的製備.......................................................................30
3. 小量質體DNA製備.....................................................................30
4. 中量質體DNA製備.....................................................................30
5. 大量質體DNA製備.....................................................................31
6. 洋菜膠中DNA片段回收(Gel Elution) .......................................31
7. 逆轉錄反應(Reverse transcription；RT) ......................................31
8. 聚合酵素鏈鎖反應(Polymerase Chain Reaction；PCR) ............32
9. 即時聚合連鎖反應(Real-Time PCR) ..........................................32
10. EGFP 與siRNA 載體接合反應(Ligation) ................................32
11. 質體轉殖(Transformation) .........................................................33
12. 慢病毒(Lentivirus production) ...................................................33
13. 測試病毒效價(virus titer) ..........................................................34
14 西方點墨法(Western Blot) ..........................................................35
15. 免疫細胞螢光 (Immunofluorescence) 染色...........................37
16. 細胞培養(cell culture )方法......................................................38

第三章 結果
1. 經由建立Sox1 siRNA knockdown的人類胚胎幹細胞探討早期神經
分化
(1)製備表現Sox1 siRNA的偽慢病毒(pseudo-lentivirus) ..............40
(2)利用表現Sox1 siRNA 的胚胎幹細胞探討其對胚胎幹細胞特
性的影響.........................................................................................41
(3)利用帶有Sox1 siRNA的細胞探討Sox1基因在神經分化的過程
中的角色.......................................................................................42
2. 利用Single cell來探討細胞的基因表現.......................................45

第四章 討論.......................................................................................48
第五章 參考文獻...............................................................................56
圖表.......................................................................................................65

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