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系統識別號 U0026-0702201409592900
論文名稱(中文) 使人類誘導型多潛能幹細胞分化成血管內皮細胞之起始驅動因子
論文名稱(英文) Essential drivers for the differentiation of endothelial cells from human induced pluripotent stem cells
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 102
學期 1
出版年 103
研究生(中文) 陳育瑞
研究生(英文) Yu-Reui Chen
學號 s96004022
學位類別 碩士
語文別 中文
論文頁數 70頁
口試委員 指導教授-江伯敏
口試委員-陳芃潔
口試委員-蔡坤哲
中文關鍵字 iPS 細胞  血管內皮細胞  分化  訊號刺激 
英文關鍵字 iPS  endothelial cells  differentiate  essential signal 
學科別分類
中文摘要 血管內皮細胞不論是在組織的修復跟器官的重組都扮演著很重要的生理
功能,但是如何利用簡單又有效的方法去得到從iPS 細胞分化而來的血管內皮
細胞卻仍未知,因此我們想要嘗試去建造一個能讓iPS 細胞分化成為血管內皮
細胞的完整系統,並利用這個系統去解釋是哪些訊號的刺激去控制整個血管內
皮細胞的分化。
而我們發現,血管前驅細胞跟成熟血管細胞可以在完整定義的系統下,藉
由人工化合物以及重組蛋白的刺激而有效的形成。另外,BMP4 在從血管前驅
細胞分化為血管內皮細胞的形成上是必需的細胞激素。透過利用shRNA
knockdown 特定的基因之後,我們發現在血管前驅細胞分化為血管內皮細胞過
程中,無論是細胞或是細胞表面之間都需要特定的分子或媒介來完成這個過程,
而這個過程也都與BMP4 的訊息傳遞路徑有所關聯。
具有使血管內皮細胞高效率分化且不需要任何含動物成分的系統讓我們
對於臨床治療的領域又往前邁一大步。除此之外,透過我們所定義的系統來調
控單一因子,我們可以證明並解釋在從血管前驅細胞分化成為血管內皮細胞的
過程裡,驅使整個分化主要的核心系統為何。
英文摘要 It is important to develop a way to obtain endothelial cells from induced
pluripotent stem (iPS) cells. The endothelial cells can be used for tissue repair,
organ engineering and biological characterizations. However, an efficient and clean
method to obtain endothelial cells from induced pluripotent stem (iPS) cells is still
absent. Here we tried to develop a defined system to differentiate iPS cells into
endothelial cells and use the system to further elucidate the signals essential for the
formation of endothelial cells.
We found endothelial precursors and mature endothelial cells can be derived
efficiently in the defined system composed of only synthetic chemicals and
recombinant proteins. Further, BMP4 is the essential trigger for the formation of
endothelial cells from hemangioblasts. By knocking down gene expression with
shRNA, we demonstrated a specific and selective set of intracellular and
cell-surface mediators required for the hemangioblasts-to-endothelium transition.
The high-efficiency derivation of endothelial cells by the defined system
allows us to rapidly extend our finding to the therapeutics. Further, by manipulating
individual factors and mediators during differentiation, we demonstrate the potential
to decipher core programs driving the formation of endothelial cells in the near
future.
論文目次 INTRODUCTION............. 1
再生醫學-臨床研究的的新道標 ......... 1
胚胎幹細胞與再生醫學 .......... 2
誘導型多功能幹細胞-劃時代的新技術 ....... 3
誘導型幹細胞的分化可能性 .......... 4
何謂血管內皮細胞? .......... 5
血管內皮細胞是組織與器官修復的主要角色 ...... 6
血管內皮細胞與其前驅細胞的研究 ........ 7
血管內皮細胞的前驅細胞-成血管血球前驅細胞 ..... 8
驅使中胚層細胞發展的起始因子 ........ 9
從中胚層細胞行成血管內皮細胞 ........ 10
BMP4 對於血管內皮細胞的發展與生理功能 ....... 11
BMP4 的功能與歷史 ........... 11
血管內皮細胞行成中的Notch 訊息傳遞路徑 ....... 12
關於血管內皮細胞成長的重要轉譯因子 ...... 13
ETV2:血管內皮細胞形成的起始訊號 ....... 14
血管內皮細胞早期的調控者:Fli1,GATA2 和 LMO2 ..... 15
V
Fox 蛋白家族 :調控血管形成的因子 ....... 16
另一種簡單且直接的分化 :轉分化 ....... 17
總結 ............. 18
MATERIAL AND METHODS .......... 20
Human iPS and ES cell culture ......... 20
Human mesodermal cell differentiation & culture ..... 20
Human endothelial cells differentiation & culture ..... 20
Capillary assay ............ 21
Immunofluorence stain ........... 21
Live tracing image ............ 21
Whole scan image ........... 22
Flow cytometry ........... 22
Transfection for virus production ......... 22
Virus concentration ........... 23
Transduction & antibiotic selection ........ 23
Statistical analysis ........... 23
RESULT ............. 24
藉由Wnt agonist,activin,和FGF 驅使人類iPS 細胞分化為中胚層細
胞,其中包含有表達KDR 的成血管血球前驅細胞 .... 24
VI
KDR(+)的成血管血球前驅細胞進一步分化成血管內皮細胞 ... 25
具功能性的血管內皮細胞會形成網狀微血管 ...... 27
BMP4 是主要驅使血管內皮細胞形成的因素 ...... 27
BMP4 的訊息傳遞路徑會經由SMAD1 以及SMAD5 調控並影響目標
基因 ............. 28
NOTCH、ERG 以及GAT2 是血管內皮細胞生成的重要轉譯因子 .. 29
DISCUSSION ............ 31
影響血管內皮細胞分化的微環境因素 ........ 31
老鼠誘導幹細胞與人類模型的差異 ........ 33
BMP4 經由SMAD1 傳遞分化的訊息 ....... 34
NOTCH 信號對於血管內皮細胞形成的可能性 ..... 35
shRNA knock-down 的限制 ......... 36
ID1 與GATA2 在血管內皮細胞分化的關係 ...... 37
ERG 在血管內皮細胞分化中的角色 ....... 37
傳遞分化的訊息是獨立存在還是彼此交互作用? ...... 38
藉由表達特定基因達到“Transdifferentiation”的效果 ..... 39
FIGURE ............. 41
Figure 1. 中胚層中有表達KDR 的細胞-成血管血球前驅細胞 .. 41
Figure 2. 從iPS 細胞分化形成血管內皮細胞 .... 43
VII
Figure 3. 成熟血管內皮細胞形成網狀微血管 .... 48
Figure 4. BMP4 是最重要的分化因子之一 ...... 50
Figure 5. 藉由shRNA knock down 的方式去探索BMP4 如何調控血
管內皮細胞的分化 .......... 53
Figure 6. Knocking down 血管內皮細胞專屬的轉譯因子去探索主要
的起始分化因子........... 56
TABLE ............. 59
Table 1. 人工計數低密度細胞培養中的血管內皮細胞數目 ... 59
Table 2. Total shRNA target ......... 60
REFERENCE ............ 61
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