臺灣博碩士論文加值系統

胚胎幹細胞擁有在體外培養時自我增生，以及可以分化為全身各種細胞型態的能力，因此被認為是可使用於再生醫學及發育研究的最佳素材。此外，人類胚胎幹細胞的基因修改是很重要的工具，其可用於基因功能探討、特定細胞的純化以及移植後的細胞追蹤等。我們的實驗目的是希望建立可產生綠色螢光的人類胚胎幹細胞株，並在其分化成特定種類神經細胞時產生紅色螢光，以用於分化為特定神經細胞的純化。我們使用piggyBac轉座子系統做為鑲嵌外來基因於TW1的轉殖基因方式。在piggyBac的質體上，我們一開始使用了多種的萬能性幹細胞的標記啟動子來驅動G418抗性以及綠色螢光的報導基因，如人類Nanog和不同長度片段的人類Oct4 啟動子。我們發現近端和完整的人類Oct4啟動子可以於未分化的TW1 hESCs，驅動下游neomycin resistant gene和螢光表現。而為了獲得穩定的人類胚胎幹細胞轉殖基因效率，我們使用CAG promoter大量表現piggyBac transposase以提高鑲嵌效能。將帶有transposase和外來基因的piggyBac質體以電穿孔方式送入TW1細胞後， CAG-GFP約有40~50%的螢光表現，而Oct4-GFP的螢光表現約有18%。接著我們使用G418進行四週的抗生素篩選，觀察仍約有近25%的螢光持續表現。為獲得在神經發育的不同階段會有不同螢光表現的人類胚胎幹細胞株，我們先測試以Nestin啟動子來驅動紅色螢光報導基因的質體，並將此質體以電穿孔方式轉殖入已分化12天的TW1細胞中，或以轉染的方式轉殖入human neuroblastoma NT-2細胞中，發現以上兩者皆有紅色螢光的表現。以此為基礎，我進一步在質體Oct4-NeoGFP/pXLBac加入了以Nestin promoter或是αTubulin promoter驅動紅色螢光以標定神經幹細胞。而在將經過測試後，質體Oct4-NeoGFP/Nestin-DsRed/pXLBac可成功在TW1細胞中有綠色的螢光表現
，在NT-2細胞中有紅色的螢光表現；但質體Oct4-NeoGFP/αTubulin-DsRed/pXLBac則在TW1細胞中同時有紅色和綠色螢光表現。我們希望能以此方式產製各種hESC細胞株，以應用在特定細胞純化、基因功能表現的研究、以及細胞移植等生物醫學研究。

The derivatives of pluripotent embryonic stem cells (ESCs) are considered to be functional and unlimited cell sources for basic developmental studies and clinical regenerative medicine. Genetic targeted human embryonic stem cells will further benefit the exploring of gene function, cell purification, and the cell tracking after transplantation into the tissues. Our purpose is to generate genetic modified hESCs for marking undifferentiated ESCs and specific lineage derivatives. The newly-developed Piggybac transposon system is applied to enhance the DNA integration efficiency in hESCs. We initially observed that 40-50 % TW1 hESCs were transfected using pCAG-GFP plasmid with Amaxa electrotransfection reagents. Several promoter regions, including the human Nanog and different segments of human Oct4 promoter, were cloned into a locus upstream the fused GFP and neomycin resistant genes. Our data revealed that only the proximal and full human Oct4 promoter constitutively drove the GFP expression and rendered the transfected cells survived for 4 weeks after G418 selection. The reporter construction for neural stem cells and mature neural cells, Oct4-NeoGFP/Nestin-DsRed/pXLBac and Oct4-NeoGFP/αTubulin-DsRed /pXLBac, respectively, were electroporated into hESCs and human neuroblastoma NT2 cells for the validation of fluorescent protein expression at specific neural stages. The harvest of the labeled specific neural cells will be applied for biomedical researches, such as developmental studies, functional cell analysis and cell transplantation.

目錄

中文摘要 i
Abstract ii
目錄 iii
前言 1
一、 人類胚胎幹細胞分化為神經幹細胞的研究與發展 1
二、 人類胚胎幹細胞的培養、繼代 1
三、 人類胚幹細胞的轉基因和K-I研究 2
四、 本次實驗主要內容與設計 3
實驗材料與方法 5
一、 儀器與材料 5
(一) 實驗儀器與耗材 5
(二) 試劑 5
(三) 進行轉基因人類胚幹細胞質體建構之材料 6
(四) 人類胚幹細胞株、神經前驅細胞 6
(五) 滋養層細胞株 6
二、 試驗方法 6
(一) 質體 DNA 的抽取 6
(二) DNA 聚合酶連鎖反應（polymerase chain reaction, PCR） 6
(三) PCR 產物之回收 7
(四) PCR產物純化 (PCR Purification) 7
(五) 黏接反應（ligation） 8
(六) 限制酵素剪切 (restriction enzyme digestion) 8
(七) DNA 分子端的補齊 8
(八) 以限制酵素切位進行接合反應( restriction enzyme site ligation) 8
(九) 製備大腸桿菌勝任細胞 8
(十) 大腸桿菌的化學轉型作用（chemical transformation） 9
(十一) fmoles與 ng轉換公式如下： 9
(十二) 引入片段和載體以限制酵素切位黏合之比例計算方法如下 9
(十三) 電穿孔勝任細胞的製備 9
(十四) 利用lipofectamine 2000 (invitrogen) 試劑進行DNA轉染 (fransfection) 10
(十五) 滋養層細胞(feeder layer)的製作 10
(十六) 人類胚幹細胞(hESCs)的培養 10
(十七) 人類胚幹細胞(hESCs)的繼代 11
(十八) 人類胚幹細胞(hESCs)的冷凍與儲存 11
(十九) 人類胚幹細胞(hESCs)的解凍 11
(二十) 人類胚幹細胞於mTeSRI nonfeeder環境下的繼代 12
(二十一) 人類胚幹細胞於mTeSRI nonfeeder環境下的冷凍 12
(二十二) 對人類胚幹細胞進行單細胞分離篩選 (single cell dissociation) 12
(二十三) 對人類胚幹細胞進行單細胞冷凍及解凍 12
(二十四) 人類胚幹細胞的無滋養層培養法 (non-feeder culture) 13
(二十五) 使用 Human Stem Cell NucleofectorR Starter Kits (amaxa) 進行人類胚幹細胞 DNA 轉染 13
(二十六) 人類胚幹細胞的神經分化(neural differentiation) 14
(二十七) 流式細胞儀分析操作 14
(二十八) 免疫螢光染色法 15
結果 16
一、 構築使用Nanog或不同長度的Oct4 promoter驅動表現G418抗性及綠色螢光報導基因的piggyBac轉座子載體 16
二、 TW1人類胚幹細胞株的鑑定與培養 16
三、 人類胚幹細胞的electroporation 17
四、 以G418及sorter篩選與純化以piggyBac轉座子系統產製出具綠色螢光轉基因的TW1-Oct4人類胚幹細胞 17
五、 將以Oct4 promoter驅動綠色螢光轉基因的TW1細胞進行神經分化與鑑定 17
六、 構築使用Nestin promoter驅動表現紅色螢光報導基因的piggyBac轉座子載體 18
七、 構築同時擁有以Oct4 promoter驅動表現G418抗性及綠色螢光報導基因，且以Nesin /α-Tubulin promoter驅動表現紅色螢光報導基因的piggyBac轉座子載體 18
討論 19
參考文獻 22
圖表 29

圖表目錄

表 1. piggyBac載體構築中使用之引子列表。 29
圖 1. 建立之neuron-specific-reporter 轉基因人類胚幹細胞株的質體設計示意圖。 30
圖 2.構築含由CAG promoter 驅動之G418抗性及綠色螢光蛋白DNA片段的piggyBac 轉座子質體示意圖。 31
圖 3. 構築含由Nanog promoter 驅動之G418抗性及綠色螢光蛋白DNA片段的piggyBac 轉座子質體示意圖。 32
圖 4. 構築含由Nanog-Nanog promoter 驅動之G418抗性及綠色螢光蛋白DNA片段的piggyBac 轉座子質體示意圖。 33
圖 5. 構築含由Oct4A-Nanog promoter 驅動之G418抗性及綠色螢光蛋白DNA片段的piggyBac 轉座子質體示意圖。 34
圖 6. 構築含由Oct4B promoter 驅動之G418抗性及綠色螢光蛋白DNA片段的piggyBac 轉座子質體示意圖。 35
圖 7. 構築含由Oct4 promoter 驅動之G418抗性及綠色螢光蛋白DNA片段的piggyBac 轉座子質體示意圖。 36
圖 8. TW1人類胚幹細胞株的pluripotent markers免疫螢光染色鑑定以及核型分析(karyotyping)。 37
圖 9. 人類胚胎幹細胞的培養。 38
圖 10. 人類幹細胞株以酵素Accutase打散為單細胞進行繼代培養。 39
圖 11. 將構築的質體以電穿孔方式轉殖入TW1細胞株並進行繼代培養。 40
圖 12. 將構築的質體以電穿孔方式轉殖入TW1細胞株，並利用流式細胞儀觀察轉殖基因的TW1細胞株螢光狀況。 41
圖 13. 將轉殖入Oct4-NeoGFP/pXLBac的TW1細胞，利用轉殖入的neomycin特徵，以G418做抗性篩選，以獲得Oct4-TW1轉殖細胞。 42
圖 14. 將轉殖入Oct4-NeoGFP/pXLBac的TW1做分化測試。 43
圖 15. 利用Sorter將轉殖入Oct4-NeoGFP/pXLBac的TW1做篩選蒐集。 44
圖 16. 構築含由Nestin promoter 驅動紅色螢光蛋白DNA片段的piggyBac 轉座子質體示意圖。 45
圖 17. 構築含由Oct4 promoter 驅動G418抗性及綠色螢光蛋白DNA片段，由Nestin promoter驅動紅色螢光蛋白的piggyBac 轉座子質體示意圖。 46
圖 18. 構築含由Oct4 promoter 驅動G418抗性及綠色螢光蛋白DNA片段，由αTubulin promoter驅動紅色螢光蛋白的piggyBac 轉座子質體示意圖。 47
附錄 1. 本實驗Oct4 enhancers和promoter的設計依據。 48

附錄 2. 構築中使用的DNA marker，1 Kb Plus DNA Ladder (Invitrogen™) in 0.9% agarose gel stained with ethidium bromide。 49

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