臺灣博碩士論文加值系統

胞外基質(extracellular matrix)在胚胎著床以及胚胎發育的過程當中都扮演著重要的角色，從先前的研究中指出功能性阻斷胞外基質接受器會導致胚胎著床失敗，而缺乏胞外基質則會造成胚胎致死。胞外基質以及他們的接受器�{整合素(integrins)之間的交互作用而促進細胞的移動，是胚胎著床以及胚胎發育的關鍵。在我們的研究中，我們比較不同胞外基質對於胚胎幹細胞(ES cells)和囊胚的滋養層細胞(trophoblast cells)移動的影響，接著研究下游的相關機制。我們發現第四型膠原蛋白(collagen IV)、纖維粘連蛋白(fibronectin)和層粘連蛋白(laminin)皆會表現在老鼠囊胚時期的內細胞團(inner cell mass)以及滋養層外胚層(trophoectoderm)。利用老鼠胚胎幹細胞株ESC26GJ所形成的胚胎體(embryoid body)模擬囊胚的內細胞團，並且分別培養於表面有或沒有塗敷第四型膠原蛋白、纖維粘連蛋白、層粘連蛋白、第一型膠原蛋白和matrigel的培養基上。這些不同胞外基質中，第四型膠原蛋白對於刺激胚胎體的擴張有最大的效應，即使在利用絲裂黴素 C抑制細胞的增生之下，第四型膠原蛋白依然能夠刺激胚胎體的擴張。胚胎幹細胞會表現��2和��1整合素而無��1的表現，且第四型膠原蛋白促進胚胎體擴張的效應會被RGD�戎H及��2和��1整合素的阻斷抗體所阻礙。處理細胞鬆弛素D（cytochalasin D），一種會使連結於整合素的肌動蛋白(actin)細胞骨架結構瓦解的試劑，也能夠阻斷第胚胎體在第四型膠原蛋白上的擴張效應。有趣的是我們發現當胚胎幹細胞培養於第四型蛋白質之上時，兩種上皮細胞�{間質細胞轉移(epithelial-mesenchymal transition)的標誌，纖維粘連蛋白(fibronectin)和波形蛋白(vimentin)具有向上調節的表現。除此之外，在這些不同的胞外基質之中，第四型膠原蛋白對於促進老鼠囊胚的滋養層細胞擴張也具有最佳的效應，而這種效應也能被細胞鬆弛素D抑制下來。我們的結果證明第四型膠原蛋白對於促進胚胎幹細胞和囊胚的滋養層細胞移動有最佳的效果，且第四型膠原蛋白促進胚胎幹細胞的移動是經由��2/��1整合素以及肌動蛋白細胞骨架所調節。我們的結果可以幫助用於幹細胞移植治療以及人工生殖技術的改良之上。

Extracellular matrix (ECM) plays an important role in embryo implantation and embryogenesis, since previous studies have shown that functional blockade of ECM receptors results in implantation failure and deficiency in ECM proteins causes embryonic lethality. Interaction between ECM proteins and their receptors, integrins, promotes cellular migration, which is critical for embryo implantation and development. In this study, we compared the effects of various ECM proteins on the migration of embryonic stem (ES) cells and trophoblast cells of the blastocysts, followed by examination of the underlying mechanisms. We found that collagen IV, fibronectin and laminin were expressed in the inner cell mass (ICM) and trophoectoderm of the mouse blastocysts. Using a mouse ES cell line, ESC26GJ, embryoid bodies (EB) were generated to mimic the ICM and cultured on surfaces coated with or without collagen IV, fibronectin, laminin, collagen I and matrigel. Among the various ECM proteins, collagen IV maximally stimulated EB expansion, even under mitomycin C inhibition of proliferation. ES cells expressed ��2 and ��1 integrin subunits, but not ��1 integrin. The promoting effect of collagen IV on EB expansion was prevented by the RGD peptide and blocking antibodies against ��2 and ��1 integrins. Cytochalasin D, a reagent disrupting actin cytoskeleton that links to integrins, also blocked the enhancing effect of collagen IV on EB expansion. Interestingly, we found that the expression of two epithelial-mesenchymal transition (EMT) markers, fibronectin and vimentin, was up-regulated in ES cells when culturing on collagen IV. In addition, among various ECM proteins, collagen IV also had the greatest stimulatory effect on trophoblast outgrowth of the mouse blastocysts. The enhancing effect of collagen IV on trophoblast outgrowth could be blocked by cytochalasin D. Our results demonstrated that collagen IV maximally stimulated migration of ES cells and trophoblast cells of the blastocysts and the collagen IV-promoted ES cell migration was mediated by signaling through ��2/��1 integrin and actin cytoskeleton. Our findings may help improve the successful rate of stem cell transplantation and assisted reproductive technology

中文摘要...6
Abstract...8
Chapter 1. Introduction...10
A. Early embryo development and implantation...10
B. The role of ECM and ECM receptors in embryo implantation and peri-implantation embryo development...13
C. The signal transduction between ECM and integrins...16
D. Embryonic stem cell as a model for studying embryogenesis...19
E. Purpose...20
Chapter 2. Materials & Methods...22
A. Reagents and antibodies...22
B. ES cell culture...23
C. Blastocyst isolation...24
D. EB aggregation...24
E. Measurement of EB expansion and trophoblast outgrowth on different ECM substrata...25
F. Cell survival analysis...26
G. Lactate dehydrogenase activity measurement...27
H. Immunocytochemistry...27
I. Immunoblotting...29
J. Statistical analysis...31
Chapter 3. Results...32
A. Expression of ECM in blastocyst...32
B. Effects of various ECM on EB expansion...32
C. Effects of different ECM on EB expansion under mitomycin C inhibition of cell proliferation...34
D. Roles of integrins in collagen IV-promoted EB expansion...36
E. Roles of actin cytoskeleton in collagen IV-promoted EB expansion...38
F. Collagen IV may turn on epithelial-mesenchymal transition in ES cells...38
G. The outgrowth of trophoblasts on different ECM..39
H. Effect of RGD peptide on trophoblast outgrowth..40
I. Roles of actin cytoskeleton in collagen IV-promoted trophoblast outgrowth...41
Chapter 4. Discussion...42
A. Effect of ECM on the migration of ES and trophoblasts cells...42
B. Function roles of ECM in the blastocyst...43
C. Use of ES cell line to study migration of embryonic cells...44
D. Mechanisms underlying enhanced ES cell migration on ECM...45
E. The role of ECM in trophoblasts migration...47
F. Collagen IV may facilitate stem cell transplantation and assisted reproductive technology...48
References...76

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