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研究生:廖耕含
研究生(外文):Kang-Han Liao
論文名稱:探討Zfx調控小鼠胚幹細胞的自我更新機制
論文名稱(外文):Investigation of the role of Zfx in Self-Renewal of Mouse Embryonic Stem Cells
指導教授:沈家寧
指導教授(外文):Chia-Ning Shen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系暨研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:46
中文關鍵詞:幹細胞zfxtcloct4nanog自我更新
外文關鍵詞:stem cellzfxtcloct4nanogself-renew
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胚幹細胞藉由細胞激素(cytokine)和生長因子(growth factor)的調控來維持其多潛能分化特性(pluripotency)及自我更新(self-renewal)的生長,最近的研究指出鋅手指蛋白X(zinc-finger protein, X-linked, Zfx)參與調控胚幹細胞的自我更新,而在Zfx剃除的實驗也指出第一型T細胞白血病因子(Tcl-1)可能是Zfx基因所調控的下游目標基因,而Tcl-1也在穩定活化Akt訊息傳遞路徑上扮演重要角色,因此本論文嘗試分析Zfx是否透過調控Tcl-1的基因表現及活化Akt訊息傳遞路徑來調控胚幹細胞的自我更新。首先我們發現抑制PI3K/Akt的訊息傳遞路徑會降低Nanog、Oct4及Tcl-1的表現,但是並不會改變影響Zfx;而在胚幹細胞過量表現Zfx,也並不會影響Tcl-1的表現,因此我們推測Tcl-1的表現不是由Zfx所直接調控的。然而我們觀察到在胚幹細胞過量表現Tcl-1會提升Oct4基因的表現,根據目前研究指出,Tcl1為Oct4基因下游的分子,我們推測Tcl-1扮演回饋調控Oct4基因的角色。此外本論文建立誘導式ShRNA的表現系統以在特定時點抑制Zfx的表現,發現Zfx抑制不直接影響Oct4和Nanog的表現,也不影響自我更新下小鼠胚幹細胞的生長。推測Zfx可能透過其他訊息傳導路徑來調節胚幹細胞的自我更新。
Embryonic stem cells (ESCs) possess the ability to undergo multiple self-renewal cycles whilst maintaining their pluripotent state. The pluripotency and self-renewal of ESCs are regulated by a variety of cytokines/growth factors. Recent study showed that the zinc-finger protein-Zfx impaired the self-renewal of ES cells. It was indicated that T-cell leukimia 1 (Tcl-1) could be the downstream effector of Zfx. Thus we initially attempted to determine whether Tcl-1 is directly regulated by Zfx though AKT pathway. First, we showed that inhibit PI3K/Akt pathway would downregulate Nanog, Oct4, and possibly Tcl-1, but not Zfx. Addition, overexpression of Zfx did not affect the level of Tcl-1 in ESCs indicating that Tcl-1 might not be a direct target of Zfx. However, we demonstrated showed overexpression of Tcl-1 indeed upregulated the level of Oct4 indicating a feedback role of Tcl-1 in maintenance of ESC pluripotency. We also confirmed reduced level of Zfx did not affect expression level of Oct4 and Nanog, and the self-renewal growth of mouse ESCs by condition knockdown system with ShRNA. Taken together, Zfx may regulate self-renewal of embryonic stem cells though other pathway.
中文摘要-------------------------------------------------1
ABSTRACT-------------------------------------------------2
I. INTRODUCTION-----------------------------------------3
I.1 Mouse embryonic stem cells--------------------------3
I.2 Mechanisms to maintain ES cell self-renewal and
pluripotency----------------------------------------3
I.3 Zinc finger protein, X-linked (ZFX)-----------------6
I.4 PI3K/AKT signaling pathway--------------------------9
I.5 Aims------------------------------------------------10
II. MATERIALS AND METHODS-------------------------------11
II.1 ES cell culture------------------------------------11
II.1.1 Isolate primary mouse embryonic fibroblasts (MEF)11
II.1.2 MEF culture medium-------------------------------11
II.1.3 MEF culture condition----------------------------11
II.1.4 R1 Strain----------------------------------------12
II.1.5 ESCs culture medium------------------------------12
II.1.6 ESCs culture condition---------------------------12
II.2 Overexpression Zfx and Tcl-1-----------------------13
II.2.1 Zfx and Tcl-1 construct with EGFP maker.---------13
II.2.2 Transfection and analysis cells------------------13
II.3 Lentivirus production------------------------------14
II.4 RNA isolation--------------------------------------14
II.4.1 Reverse Transcription----------------------------14
II.4.2 PCR----------------------------------------------14
II.4.3 Cells-to-cDNA™ II Kit (Ambion company)-----------15
II.5 Establishment of conditional knockdown system------15
II.5.1 Origin of shZfx clones---------------------------15
II.5.2 Real-time PCR------------------------------------15
II.5.3 ShRNA primer design------------------------------16
II.5.3 Infection ES and selection of shZfx of ES cell line
-------------------------------------------------16
II.6 Inhibitors treatment-------------------------------16
II.7 Immunofluorescent staining-------------------------17
II.8 Western blot---------------------------------------17
II.9 HTNCre purify--------------------------------------18
III. RESULTS--------------------------------------------19
III.1 Expression of Zfx in R1 ESCs----------------------19
III.2 Zfx is not the downstream target gene of PI3K/Akt
pathway.------------------------------------------19
III.3 Modulating expression of Oct4 with Tcl-1
overexpression.-----------------------------------20
III.4 Conditionally knockdown of Zfx in R1 ESCs---------20
III.4.1 Identification of efficient shRNA constructs which
can knockdown Zfx expression in mouse embryonic
fibroblast cells.-------------------------------20
III.4.2 Conditional knockdown pSico system--------------21
III.4.3 Conditional knockdown of Zfx affect the colony
morphology and self-renewal growth of ES cells.-21
IV. CONCLUSION------------------------------------------23
V. DISCUSSION-------------------------------------------24
VI. FIGURE LEGENDS AND TABLES---------------------------27
VII. REFERENCES-----------------------------------------42
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