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標題: | 將細胞電泳技術應用在使用機械力及/或共培養系統
誘導間葉幹細胞分化之觀察 The application of cell electrophoresis on the analysis of differentiation of mesenchymal stem cells induced by mechanical stress and/or co-culture system |
作者: | I-Chi Lee 李亦淇 |
指導教授: | 楊台鴻(Tai-Horng Young) |
關鍵字: | 前十字韌帶細胞,間葉幹細胞,細胞電泳,機械拉力,共同培養系統, anterior cruciate ligament (ACL),Mesenchymal stem cells (MSCs),cell electrophoresis,mechanical stress,co-culture system, |
出版年 : | 2006 |
學位: | 博士 |
摘要: | 前十字韌帶,位在膝關節的關節腔內,對於膝蓋的穩定性相當重要。然而不幸的,它也是最容易受傷的關節腔內韌帶。目前在臨床上的治療方式是利用組織重建的方法,包含自體移植、異體移植及異種移植,或是以人造材料取代。然而這些外科手術的替代方法均無法提供長期使用上的滿足。因此,前十字韌帶的高受傷比率、非常低的自我修復率及目前臨床上處理方法的限制均成為促使此研究進入韌帶組織工程階段的動力。間葉幹細胞可以分化為多種非血球族系的細胞,包含骨頭細胞、軟骨細胞及韌帶細胞等。即使在老化的個體中,骨髓仍然易於取得並且仍能包含生物合成的活化前驅物及多重分化的細胞。此研究包含韌帶組織工程的以下幾個部分,分離及鑑定前十字韌帶細胞及間葉幹細胞;找尋合適的機械拉力以促進間葉幹細胞中與前十字韌帶細胞的細胞外間質相關之基因表現;提供合適的生理調節訊號誘導間葉幹細胞分化為韌帶細胞。
在第一章中,我們簡介了間葉幹細胞的發展及其特性,此外,我們也介紹了前十字韌帶組織工程之發展與當今待需解決之問題。 韌帶具有獨特的分子、結構及機械等複和性質,然而,卻沒有一個獨特的標記可以用來區分韌帶及其他組織。因此,發展一個良好的技術以用來區分前十字韌帶及間葉幹細胞在前十字韌帶的組織工程發展上是相當重要的。在第二章中,本研究之目的之一即是區分間葉幹細胞及前十字韌帶細胞,以在之後應用於將間葉幹細胞誘導分化為前十字韌帶細胞之鑑定。令人驚訝的,細胞電泳可以成功的區分間葉幹細胞與前十字韌帶細胞。雖然許多傳統方法例如細胞流式儀,細胞免疫化學染色,及RT-PCR等方法已被廣泛用來觀測不同細胞的特徵蛋白或基因表現,這些方法卻無法用在區分間葉幹細胞及前十字韌帶細胞。此外,這些傳統的鑑定方法不但耗費金錢且必須浪費許多時間。細胞電泳可量測細胞的移動性,我們認為可用來觀測間葉幹細胞及前十字韌帶細胞的表面電荷的差異。雖然細胞電泳無法用來觀察單一特性蛋白的表現,卻可以反應出細胞膜表面的淨電荷密度,而其變化則會受到週邊膜蛋白之遊離官能基的影響而發生改變。因此我們認為,由於細胞電泳易於操作,可以視為一個輔助細胞鑑定的有用工具。 間葉幹細胞分化到下游細胞的途徑受到多重因素的影響,例如生長因子的添加,細胞激素的分泌及賀爾蒙等。機械力已被證明對於韌帶、骨頭及軟骨的傷口癒和及重建有所影響。許多文獻已探討機械力在多種細胞所發生的效應,然而,卻鮮少有機械力對於間葉幹細胞分化的討論。 在第三章中,為觀察機械力的刺激與間葉幹細胞分化趨勢之間的關連性,我們觀察了在未添加生長及誘導分化等因子的狀況下,間葉幹細胞在3%及10%兩種拉伸條件下,骨頭、肌腱韌帶、及軟骨等三種細胞的細胞標記的表現。結果顯示兩種機械拉力均無法誘導間葉幹細胞分化為軟骨細胞。然而,在較低的拉力下骨頭系的標記均會被向上調節,而肌腱/韌帶系的標記則在較高的拉力條件時被向上調節。因此,第三章的研究顯示出機械拉力是個有效的因子可用來刺激間葉幹細胞往骨頭或肌腱/韌帶發展的早期分化途徑。 再者,雖然誘導間葉幹細胞往多種細胞分化的機制已經相當清楚,誘導間葉幹細胞往韌帶分化的機制卻仍不明確。將間葉幹細胞誘導成韌帶細胞會受到多重因素的影響,過去的文獻指出機械拉力對前十字韌帶在受傷後的復原及重建有明顯的效應,且也有文獻指出共同培養系統可將間葉幹細胞誘導成不同的下游細胞。因此,在第四章中,我們觀察了間葉幹細胞在三組誘導分化系統中,韌帶細胞的主要組成的細胞外基質-型一膠原蛋白,型三膠原蛋白及tenascin-C等基因表現的改變。此外,延續第二章的研究,我們也用細胞電泳的技術及觀察間葉幹細胞蛋白的基因表現來分析在三組誘導分化系統下,間葉幹細胞的分化行為。在此研究中,第一組誘導分化系統為將間葉幹細胞與前十字韌帶細胞進行共同培養;第二組誘導分化系統為施予機械拉力於間葉幹細胞;第三組誘導分化系統為將間葉幹細胞與前十字韌帶細胞進行共同培養後再施予機械拉力。 結果顯示出在與前十字韌帶細胞的共培養系統中,前十字韌帶會釋放出特殊的調節訊號可以促進間葉幹細胞往韌帶細胞分化,而機械力則會促進韌帶主要細胞外基質的基因表現。此外,若結合機械力的刺激及共養系統的效應則有助於輔助前十字韌帶組織工程中的復原與重建。再者,此研究也顯示出細胞電泳確實可用來觀察細胞的分化行為。因此,本研究的分析對於將間葉幹細胞用於前十字韌帶組織之修復與重建的組織工程上將有相當大的幫助。 The anterior cruciate ligament (ACL), an intraarticular ligament of the knee, is important for knee stabilization. Unfortunately, it is the most commonly injured intraarticular ligament. So far, the therapeutic options to repair torn ligaments are tissue reconstruction using autograft or allograft, reparation alone or with augmentation, or replacement using a synthetic prosthesis. Unfortunately, none of these surgical alternatives provides a long-term adequate solution. Therefore, the high incidence of ACL failures, lack of capacity for self-repair, and limitations of current treatment options have driven the research into ligament tissue engineering as a new option. Mesenchymal stem cells (MSCs) can differentiate into multiple non-hematopoietic cell lineages, including osteoblasts, chondrocytes, and ligament cells. Even in older individuals, bone marrow stroma is relatively easily harvested and contains biosynthetically active precursors and multipotent cells. This study proceed with the tissue engineering of ACL involving the isolation and identification of ACL cells and MSCs, finding the appropriate mechanical stretch to promote the up-regulation of ACL cells’ ECM gene expression of MSCs, and provide the necessary biophysical regulatory signals to induce MSCs differentiation. In Chapter 1, we introduce the development and characteristic of MSCs, besides, the progress and the problems of ACL tissue engineering are also introduced. Ligaments have a unique combination of molecular, structural, and mechanical properties, but there is no single unique marker that can be used to distinguish between ligaments and other tissues. Therefore, the development of a useful technique to discriminate ACL cells from MSCs is very important in ACL tissue engineering. In Chapter 2, the purpose of this chapter is to identify the difference between MSCs and ACL cells for the application of distinguishing theses two types of cells during the process of MSCs differentiating into ACL cells. Surprisingly, cell electrophoresis could distinguish MSCs from ACL cells successfully. Although various traditional methods such as flow cytometry, immunocytochemistry, and RT-PCR have been developed to determine the expression of specific proteins or genes to characterize different cells, they cannot be used to distinguish MSCs and ACL. In addition, using traditional methods to identify cells is not only expensive but also time consuming. Cell electrophoresis, measuring the electrophoretic mobility (EPM) of cells, is proposed to investigate the discrepancy of surface charge property of MSCs and ACL cells. Although cell electrophoresis cannot be used to determine the specific surface protein, EPM can reflect the net surface charge density of cell membrane, which can be influenced by the dissociation of functional groups of peripheral membrane proteins. It is suggested that cell electrophoresis, while simple in manipulation, can serve as a useful research tool to assist cell identification. Differentiation of MSCs into different kinds of cells is regulated by many factors, such as growth factors, cytokines, and hormones. Mechanical stretch has been shown to affect the healing and remodeling process of ligament, bone, and cartilage. Many articles have been published which discussed the effects of mechanical stretch on various cell types, however, little is known about the effects of mechanical stretch on differentiation of MSCs. In Chapter 3, in order to determine the correlation of mechanical stimulation and differentiation tendency in vitro, three groups of cell markers, bone, tendon/ligament and cartilage, are elongated on 3% and 10% using the Flexcell stress system, without addition of other growth and differentiation factors. The results reveal that mechanical stretch couldn’t induce MSCs into cartilage cell lineage. In contrast, the bone cell lineages markers are up-regulated at strains of low magnitudes and the tendon/ligament cell lineages markers are up-regulated at high magnitudes strain. Therefore, this study shows that mechanical stretch is an effective factor that could regulate the early stage differentiation pathway of MSCs into bone or ligament/tendon cell lineages. Furthermore, since most of the mechanisms of induction MSCs to desire cells are clearly understood, the induction mechanism of MSCs to ACL cells is still not obvious. Differentiation of MSCs into ACL cells is regulated by many factors. Since mechanical stress affects the healing and remodeling process of ACL after surgery significantly, co-culture system had also showed the promise to differentiate MSCs toward different kinds of cells on current research. In Chapter 4, we investigate the gene expression of major extracellular matrix component molecules of ACL cells, collagen type I, type III, and tenascin-C of MSCs under three induction groups. In addition, to follow the study on chapter 2, cell electrophoresis technique and mRNA level gene expression of MSC protein are also used to analyze the differentiation of MSCs. Group I is the MSCs co-culture with ACL cells. Group II is the MSCs exposure to mechanical stress. Group III is the MSCs exposure to mechanical stress after co-culture with ACL cells. The results reveal that specific regulatory signals releasing from ACL cells appear to be responsible for supporting the selective differentiation toward ligament cells in co-culture systems and mechanical stress promotes the secretion of key ligament ECM components. In addition, the results also reveal that combined regulation of mechanical stress and co-culture effect could assist the development of healing and remolding of ACL tissue engineering. Furthermore, this study also demonstrates that cell electrophoresis could be used in investigation of cell differentiation. Also, analysis of the data suggests the feasibility of utilizing MSCs in clinical applications for repairment or regeneration of ACL tissue. |
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