博碩士論文 982204010 詳細資訊




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姓名 簡嘉瑩(Chia-ying Chien)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 建立安全的誘導性幹細胞的重新編寫方法
(Developing a reprogramming method for generating safe induced pluripotent stem cells)
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摘要(中) 幹細胞分化成各種型態細胞的能力稱之為多能性,但終端分化的體細胞則失去了這些多能性。在近年的研究中發現,將體細胞大量表現特定的幹細胞轉錄因子 (Yamanaka 因子, YFs) ,可使細胞重新編寫(reprogram)成具有多能性的細胞,這些即所謂的誘導性幹細胞 (iPSC)。然而YFs通常是透過反轉錄病毒介導方式送入,細胞中大量表現,導致基因體的嵌入現象,引發插入性突變嚴重的關切。隨著iPSC研究的進步,漸漸發展出許多安全性誘導iPSC產生的reprogramming方式,其中像是(1) 利用具有可回復的短暫通透性細胞膜之細胞,再加入ES 細胞的萃取液培養,(2) 或是利用帶有細胞通透序列的YFs重組蛋白,來誘導iPSC的產生。雖然這兩種方式避免了基因體的嵌入性突變,但其產生iPSC的效率非常低。因此我們想要利用結合這兩種方式,將具有通透性的體細胞同時加入帶有細胞通透序列的YFs重組蛋白,來增加iPSC的產生效率。首先,建立攜帶由Oct4啟動子驅動的報導基因Oct4-puromycine 或 Oct4-luciferase-IRES-GFP的穩定纖維母細胞株,接著利用鏈球菌溶血素 (SLO) 將細胞膜產生短暫通透性,最後再加入YFs重組蛋白促使細胞reprogram並產生多能性,並且檢測puromycin或是GFP的表現篩檢reprogramming的能力。現階段利用細菌系統表現的GST-Oct4、GST-Nanog、Sox2 LpRH、Lin28 LpRH 和 c-Myc LpRH蛋白,同時加入小分子物質 (VPA、BIX-01294和 EVR) 來誘導通透性纖維母細胞的多能性,目前已進行數次reprogramming 的實驗,但成功的比率偏低。未來希望可利用昆蟲系統表現YFs重組蛋白,增加具有轉譯後修飾作用YFs。如果成功誘導iPSC產生後,可再進一步測定細胞的多能性,確認是否會產生類似ES cell的colony現象,以及判定內部的基因表現情形,或是胚胎體 (embroid body)形成的分化測試。
摘要(英) Stem cells can differentiate into most cell types while differentiated somatic cells have lost this pluripotency.  Recent studies have shown that somatic cells can be reprogrammed by over-expression of defined transcription factors (Yamanaka factors, YFs) to become pluripotent stem cell (called as induced pluripotent stem cells, iPSC).  Over-expression of YFs is usually mediated by retrovirus and whose integration into genome raises serious concern about the insertional mutagenesis.  To develop safer iPSC, one reprogramming method has incubated reversibly permeable target cells in ES cell extracts and another has used recombinant cell–penetrating YFs proteins to generated iPSC.  Although these two methods avoid insertional mutagenesis, unfortunately, they generate iPSC with only low efficiency.  Therefore, we like to combine these two approaches by treating somatic cells with permeable system and cell-penetrating YFs proteins simultaneously to increase the reprogramming efficiency.  Firstly, stable clones of fibroblasts carrying Oct4 promoter driven puromycine or IRES-GFP reporter genes were established, then, they were treated with streptolysin-O (SLO) to make their plasma membrane transiently permeable.  Finally, recombinant YFs proteins will be added to reprogram them to pluripotent state and the reprogramming efficiency will be examined by puromycin or GFP selection.  Currently, the pluripotency of permeable fibroblasts were induced by bacterially expressed GST-Oct4、GST-Nanog、Sox2 LpRH、Lin28 LpRH and c-Myc LpRH proteins in the presence of small compounds (VPA、BIX-01294 and EVR). Unfortunately, the reprogramming efficiency is rather low.  In the future, YFs proteins will be expressed by insect cells that allow post-translational modification of these factors.  Once iPSC clones have been generated, their pluripotent state will be examined by ES-like colony formation, gene expression profiling assays, and embryoid body formation.
關鍵字(中) ★ 重新編寫
★ 誘導性幹細胞
★ 重組蛋白
★ 細胞穿透序列
★ 短暫通透性細胞膜
★ 鏈球菌溶血素O
關鍵字(英) ★ streptolysin-O
★ plasma membrane transiently permeable
★ cell penetrating peptides
★ recombinant proteins
★ iPSC
★ reprogramming
論文目次 中文摘要...............................................i
英文摘要 (Abstract)...................................ii
聲明 (Declaration)...................................iii
誌謝..................................................iv
目錄...................................................v
圖表目錄............................................viii
一、緒論...............................................1
1 胚胎發育 (embryogenesis)............................1
2 幹細胞 (Stem cell),,,,,,............................3
3 胚胎發育重新編程 (Reprogramming)....................5
4 研究動機與目的......................................9
二、實驗材料與方法....................................10
1 細胞株.............................................10
2 菌株...............................................10
3 穩定細胞株.........................................10
3-1 帶有Oct4 promoter的C3H10T1/2穩定細胞株...........10
3-2 帶有Neomycin抗性基因的Feeder cell................10
3-3 帶有puromycin及neomycin抗性基因的Feeder cell.....11
4 質體的建構.........................................11
4-1 穩定細胞株之質體建構.............................11
4-1-1 pPuro Oct4 promoter............................11
4-1-2 pStable IhrG Oct4 P............................11
4-2 E. coli中表現蛋白質之質體建構....................12
4-2-1 pQE60 LpRH-YFs.................................12
4-2-2 pET32a-YFs-LpRH................................12
4-3 質體建構的酵素作用...............................12
4-3-1 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 12
4-3-2 PNK (Polynucleotide kinase, NEB)...............13
4-3-3 Klenow (DNA polymerase I, Large Fragment, NEB).14
4-4 CIP (Calf Intestinal Alkaline Phosphatase, NEB)..14
4-5 Vector DNA 的製備................................14
4-6 Insert DNA的製備.................................14
4-7 DNA 接合作用 (ligation)..........................14
4-8 大腸桿菌的轉型作用 (Transformation)..............15
4-9 小量純化並篩選質體DNA............................15
5 細胞株的轉染作用 (transfection)....................15
5-1 轉染之細胞株培養.................................15
5-2 轉染液製備.......................................15
6 誘導YFs-LpRH protein的表現與純化...................16
6-1 蛋白質的誘導表現.................................16
6-2 蛋白質的純化.....................................16
6-3 蛋白質的濃縮與透析...............................18
7 細胞膜的短暫通透性 (transient permeabilization and protein treatment) ....................................19
7-1 Streptolysin O酵素活化...........................19
7-2 使細胞產生通透性.................................19
8 質體轉染誘導細胞Reprogramming......................20
8-1 將10T1/2 pStable IhrG Oct4 P細胞均勻種到6 well dish中約七分滿,使細胞貼附一天後的第2天進行質體轉染。.......20
9 西方墨點實驗 (Western blot)........................20
9-1 total lysate 的製備..............................20
9-2 誘導表現蛋白的純化過程...........................21
9-3 SDS-polyacrylamide Gel Electropheresis...........21
9-4 Blocking以及Antibody辨識.........................21
9-5 Striping.........................................22
10 啟動子活性分析 (promoter assays)..................22
三、實驗結果..........................................24
1 建構帶有Oct4 promoter調控選擇性標誌 (selection marker) 表現的穩定細胞株......................................24
2 確認穩定細胞株中是否帶有送入的質體.................24
3 利用SLO作用使10T1/2 pStable IhrG Oct4 P細胞膜產生短暫通透性..................................................25
4 架構與純化帶有蛋白質穿透序列 (cell penetrating peptides, CPPs) 的Yamanaka Factors (YFs) 重組蛋白.....25
4-1 蛋白質的誘導表現.................................25
4-2 蛋白質的純化.....................................27
4-3 用不同的培養液誘導蛋白質的表現...................29
4-4 表現純化GST-Oct4 與GST-Nanog蛋白.................29
5 將純化後的YFs-LpRH重組蛋白同時加入具有短暫性通透膜的10T1/2 pStable IhrG Oct4 P穩定胞株中..................30
6 利用plasmid transfection來誘導iPS cells的產生......31
四、討論..............................................33
1 1.9 k Oct4 promoter調控下游基因表現情形............33
2 SLO在Fibroblasts產生通透性.........................33
3 重組蛋白的純化條件與誘導表現的能力.................34
4 利用細菌表現的重組蛋白進行reprogramming的完整功能性35
5 質體DNA與小分子物質誘導iPSC的產生的可行性..........37
五、參考文獻..........................................39
圖表目錄
六、圖表..............................................46
圖一、 建構帶有Oct4 promoter調控的選擇性標誌(selection marker)的質體並送入細胞中成為穩定細胞株...............47
圖二、 檢測質體DNA是否送成功送入目標細胞中成為穩定細胞株。..................................................48
圖三、 利用SLO作用在穩定細胞株上,使細胞膜具有短暫的通透性。..................................................50
圖四、 將YFs接入表現載體的架構與菌中誘導情形。.......52
圖五、 YFs-LpRH於pET32a上的架構方式及菌中的誘導表現情形。..................................................54
圖六、 hLin28 LpRH c-Myc LpRH重組蛋白的純化。........56
圖七、 hSox2 LpRH的表現與純化。......................57
圖九、 利用western blot偵測,在加入蛋白酶抑制劑後於低溫25 ℃與16 ℃下誘導Nanog-LpRH和Oct4-LpRH誘導情形。........63
圖十、 利用SDS-PAGE偵測,在加入蛋白酶抑制劑後於低溫25 ℃與16 ℃下誘導Nanog與Oct4重組蛋白的表現情形。............64
圖十一、測試不同細菌培養液,幫助誘導蛋白質的表現。....66
圖十二、誘導純化GST-Nanog與GST-Oct4融合蛋白。.........68
圖十三、純化後的所有重組蛋白。........................69
圖十四、將純化後的重組蛋白加入具有通透性的穩定細胞株。70
圖十五、plasmid transfection誘導iPS cell產生之質體的架構。..................................................72
圖十七、利用plasmid transient transfection產生iPS cells之螢光圖。................................................75
表一、利用質體轉染細胞的條件。........................76
附錄一................................................77
圖十八、trypan blue 測試細胞通透性。..................77
圖十九、在C2C12中大量表現 Oct4 V235P。................78
附錄二................................................79
1 質體建構引子對照表.................................79
2 小分子結構.........................................79
附錄三................................................80
A 溶液及試劑配方.....................................80
B 縮寫與全名對照表-藥品及材料........................82
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指導教授 陳盛良(Shen-liang Chen) 審核日期 2012-2-1
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