臺灣博碩士論文加值系統

大規模的子宮手術會造成顯著的子宮肌肉層缺損，並可能導致嚴重的併發症，包括了日後懷孕時發生植入性胎盤及子宮破裂。最近報導顯示分離自骨髓之間質幹細胞（mesenchymal stem cells）具有自我更新的能力並可分化形成不同胚層的細胞，包括屬於中胚層的平滑肌細胞。因此，間質幹細胞應可用於強化子宮肌肉層缺損之修補，以預防子宮手術後之嚴重後遺症，然而目前仍未有相關研究。
本研究中，我們計畫運用間質幹細胞的獨特性質，以治療子宮肌肉層缺損。首先，我們研究乙型轉型生長因子（transforming growth factor-beta1）、纖維粘連蛋白（fibronectin）及壞血酸（ascorbic acid），對於誘導間質幹細胞分化形成類平滑肌細胞（smooth muscle cells）是否具有協同作用，接著再進行動物實驗，以間質幹細胞移植修復子宮肌肉層的受損。在活體外，我們發現乙型轉型生長因子、纖維粘連蛋白及壞血酸皆能促使間質幹細胞分化形成類平滑肌細胞，可從平滑肌細胞標記蛋白calponin的表現量增加看出，而這些誘導因子之促進平滑肌細胞分化效果具有協同作用。接著我們利用共同培養模式，來檢視這些經體外分化形成之類平滑肌細胞是否能抵擋滋養層細胞（trophoblast cells）的入侵，以探討其預防植入性胎盤的可能性。我們發現間質幹細胞分化形成的類平滑肌細胞相較於未分化的間質幹細胞有較強的抵擋滋養層細胞入侵能力，雖然未能達到直接分離自子宮肌肉層的平滑肌細胞其抵擋滋養層細胞入侵的能力。最後，我們建立了一個修補子宮缺損的動物實驗模式，發現於缺損處注射間質幹細胞可加速子宮缺損的修補，而且在修補完成的子宮切片偵測到注入的間質幹細胞。我們的研究結果顯示間質幹細胞可被誘導分化為類平滑肌細胞，並能應用在子宮肌肉層的修補，嘉惠接受子宮肌肉層大規模切除手術之婦女。

Extensive uterine surgery may cause significant myometrial defects that may lead to severe sequelae, including placenta accreta and uterine rupture during subsequent pregnancy. Recent evidences have suggested that mesenchymal stem cells (MSCs) derived from bone marrow are capable of self renewal and differentiation into three germ layers, including smooth muscle cells of mesodermal origin. Therefore, it is likely that MSCs may be used to strengthen the repair of myometrial defects and prevent severe sequalae following major uterine operations. However, to date there has been no study on the application of MSCs to uterine repair.
In this study, we developed an effective therapeutic alternative, utilizing the unique features of MSCs, to treat uterine defects. We investigated whether transforming growth factor-beta1 (TGF-beta1), fibronectin and ascorbic acid could synergistically induce MSCs to acquire the phenotypical characteristics of smooth muscle cells (SMCs), followed by transplantation to repair uterine defects in an animal model. In vitro, MSCs were found to differentiate to SMC-like cells in response to TGF-beta1, fibronectin and ascorbic acid, as revealed by the up-regulated expression of calponin. Furthermore, we demonstrated synergistic effects of TGF-beta1, fibronectin and ascorbic acid on MSC differentiation into SMC-like cells. Furthermore, the ability of these MSC-derived SMC-like cells to resist trophoblast invasion was examined using a co-culture model, thereby investigating their potential application to prevent placenta accreta. As compared with undifferentiated MSCs, MSC-derived SMC-like cells more efficiently resisted trophoblast invasion, though not as effectively as freshly isolated myometrial cells. Finally, we established an animal model of uterine defect repair. Injection of MSCs into the myometrial defects was shown to accelerate the repair of defects and MSCs were detected in the uterine tissue sections at the end of the repair process. Our results demonstrated that MSCs could be differentiated into SMC-like cells and utilized to repair myometrial defects, thereby benefitting women undergoing uterine surgery with extensive myometrial resection.

中文摘要...................................................6
Abstract...................................................8
Chapter 1. Introduction...................................10
A.Uterine surgery increases the risks of uterine rupture and placenta accreta in subsequent pregnancies............10
B.It is feasible to repair uterine defects by using adequately differentiated mesodermal cells from human bone-marrow-derived mesenchymal stem cell......................15
C.Purpose................................................28
Chapter 2. Materials & Methods............................29
A.Cell culture...........................................29
B.Cell treatments........................................31
C.Measurement of spheroid expansion on monolayers of various cell types........................................32
D.Cell survival analysis.................................33
E.Immunocytochemistry....................................33
F.Immunoblotting assay...................................34
G.Uterine defect model...................................35
H.MSC transplantation....................................36
I.In vivo wound healing analysis.........................36
J.Histologic examination.................................36
K.Statistical analysis...................................37
Chapter 3. Results........................................38
A.Effects of TGF-beta1 on mesodermal differentiation of MSC.......................................................38
B.Effects of fibronectin on mesodermal differentiation of MSC.......................................................39
C.Combined effects of TGF-beta1, fibronectin and ascorbic acid on mesodermal differentiation of MSC.................40
D.Ability of MSC-derived mesodermal cells to resist trophoblast invasion......................................41
E.A SD-rat model for uterine defects.....................42
F.MSC promotes uterine defect healing....................43
G.Determining whether the in vivo newly-formed myometrial cells are generated by transplanted hMSCs.................43
Chapter 4. Discussion.....................................45
A.Effects of factors other than TGF-beta1, fibronectin and AA on mesodermal differentiation of MSC...................45
B.Functional roles of MSC-derived mesodermal cells in resisting trophoblast invasion............................46
C.The mechanisms underlying the differentiation of SMCs from MSCs.................................................46
D.Determining whether the accelerated repair of myometrial defects is mediated by paracrine effects or transdifferentiation of transplanted MSCs.................48
E.Determining whether MSCs have the ability to suppress the inflammatory response.................................48
References................................................67

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