臺灣博碩士論文加值系統

唐氏症是常見的先天性遺傳疾病，主要由三染色體21 (Trisomy 21, T21) 所造成。臨床症狀主要為神經發育遲緩與智能發展不足。為了在體外研究Trisomy 21對於神經發育的影響，所仰賴的是一個可模擬神經發育的細胞模型。因此本研究主要以羊膜穿刺篩檢出具Trisomy 21 之羊水細胞(amniotic fluid-derived cells, AF cells)，藉由導入表現Oct4, Sox2, c-Myc及 Klf4四個基因，重新再程式化(reprogramming)羊水細胞後，建立具有Trisomy 21之誘導型多能幹細胞(induced pluripotent stem cells, iPS cells)。接著利用iPS cells優越的分化能力，將其分化成神經前趨細胞(neuronal progenitor cells, NPCs) 及神經細胞，進行Trisomy 21對於神經發育的影響之後續研究。由實驗結果證實，本研究可成功建立具有分化能力之Trisomy 21之iPS cells (T21 AF-iPS cells)，並且可成功將其分化為NPCs (T21 NPCs)。進一步分析這些T21 NPCs結果顯示，座落於21號染色體上之五個候選miRNA (let-7c, miR-99a, miR-125b, miR-155與miR-802)其表現量皆高於正常NPCs約1.5 倍，而受miR-155與miR-802 調控之神經發育標的基因- MeCP2則受到過度抑制而有顯著低表現。另外座落於21號染色體上的一個與阿茲海默症相關的基因amyloid precursor protein (APP)則有過度表現的現象。接著將NPCs分化成神經細胞，結果發現T21 NPCs分化成神經細胞的能力亦較差。本研究藉由羊水細胞建立之T21 AF-iPS cells，可成功在體外模擬唐氏症病患神經發育過程，且由於MeCP2在整個神經細胞發育扮演著重要角色，因此推測21號染色體上過量之miRNA表現所導致MeCP2 低表現量，可能為唐氏症病患神經發育遲緩因素之一，而過量的APP可能為唐氏症病患易罹患阿滋海默症的原因之一。

Down syndrome (DS), or Trisomy 21 (T21) syndrome, one of the most common chromosomal abnormalities, is caused by an extra duplication of chromosome 21. In studies of neuron development, experimental models based on human cells are considered to be the most desired and accurate for basic research. The generation of diseased induced pluripotent stem (iPS) cell is a critical step in understanding the developmental stages of complex neuronal diseases. Here, we generated human DS iPS cell lines from second trimester amniotic fluid (AF) cells with T21 by co-expressing Yamanaka factors (Oct4, Sox2, c-Myc and Klf4) through lentiviral delivery and subsequently differentiated them into neuronal progenitor cells (NPCs) for further analyses. T21 AF-iPS cells were characterized for the expression of pluripotent markers and for their ability to differentiate into all three germ layers by forming embryoid bodies in vitro and teratomas in vivo. The T21 AF-iPS cells maintained their unique pattern of chromosomal karyotypes: three pairs of chromosome 21. The level of amyloid precursor protein (APP) was significantly increased in NPCs derived from T21 AF-iPS cells compared with NPCs from normal AF-iPS cells. The expression levels of miR-155 and miR-802 in T21 AF-iPS-NPCs were highly elevated in the presence of low expression of MeCP2. We observed that T21 iPS-NPCs generated fewer neurons compared with controls. T21 iPS-NPCs exhibit developmental defects during neurogenesis. Our findings suggest that T21 AF-iPS cells serve as a good source to further elucidate the impairment neurogenesis of DS and the onset of Alzheimer’s disease.

中文摘要....................................................I
ABSTRACT..................................................II
ACKNOWLEDGMENTS..........................................III
CONTENTS...................................................V
LIST OF FIGURES AND TABLES..............................VIII
CHAPTER I Introduction.....................................1
1.1 Induced pluripotent stem cells.........................1
1.2 Generation of iPS cells from different strategies......3
1.3 Generation of iPS cells from amniotic fluid-derived cells......................................................4
1.4 Disease-specific iPS models............................5
1.5 Down syndrome..........................................6
CHAPTER II Motivations and Purposes of the Research........8
2.1 Selection of alkaline phosphatase positive AF-iPS cells......................................................8
2.2 Generation of T21 AF-iPS cells.........................9
2.3 The study of the neuron derived from T21 AF-iPS cells.....................................................11
CHAPTER III Materials and Methods.........................14
3.1 Selection of alkaline phosphatase positive AF-iPS cells.....................................................14
3.1.1 Cell culture of amniotic fluid-derived cells........14
3.1.2 Generation and selection of AF-iPS cells............14
3.1.3 Alkaline phosphatase and immunofluorescence staining..................................................16
3.1.4 Karyotype Analysis..................................17
3.1.5 RT-PCR..............................................17
3.1.6 In vitro differentiation.....18
3.1.7 In vivo differentiation.....18
3.2 Generation of T21 AF-iPS cells.....19
3.2.1 Cell culture and iPS cell generation.....19
3.2.2 Alkaline phosphatase and immunofluorescence staining.....20
3.2.3 Karyotype analysis.....21
3.2.4 RT-PCR and qRT-PCR.....21
3.2.5 In vitro differentiation.....22
3.2.6 In vivo differentiation.....22
3.3 The study of the neuron derived from T21 AF-iPS cells.....23
3.3.1 Neuronal progenitor cell and neuronal differentiation.....23
3.3.2 Immunofluorescence staining.....24
3.3.3 RT-PCR and qRT-PCR..................................24
3.3.4 Western blot analysis...............................25
CHAPTER IV Results........................................26
4.1 Selection of alkaline phosphatase positive AF-iPS cells.....................................................26
4.1.1 Generation of AF-iPS cells..........................26
4.1.2 Selection of fully stained AP+ AF-iPS...............26
4.1.3 Characterization of AF-iPS cells selected from feeder-free system...............................................27
4.1.4 In vitro and in vivo differentiation of AF-iPS cells.....................................................28
4.2 Generation of T21 AF-iPS cells........................38
4.2.1 Generation of iPS cells with trisomy 21.............29
4.2.2 Differentiation potential of T21 AF-iPS colonies....29
4.3 The study of the neuron derived from T21 AF-iPS cells.....................................................30
4.3.1 Differentiation of T21-iPS into neuronal progenitor cells.....................................................30
4.3.2 Hsa21-derived miRNA, gene and protein expression levels....................................................31
4.3.3 Differentiation of T21 iPS-NPCs into neuronal cells.....................................................31
CHAPTER V Discussion......................................33
5.1 Selection of alkaline phosphatase positive AF-iPS cells.....................................................33
5.2 Generation of T21 AF-iPS cells........................36
5.3 The study of the neuron derived from T21 AF-iPS cells.....................................................37
CHAPTER VI Conclusions....................................39
REFERENCE.................................................40
FIGURES...................................................48
TABLES....................................................71
PUBLICATIONS..............................................72

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