建立安全的誘導性幹細胞的重新編寫方法

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簡嘉瑩(Chia-ying Chien) 查詢紙本館藏

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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

推文