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系統識別號 U0026-2208201915435400
論文名稱(中文) 微環境調控人類骨髓間質幹細胞之肌腱分化
論文名稱(英文) Microenvironmental cues collaboratively regulate tenogenesis in human bone marrow-derived mesenchymal stem cell
校院名稱 成功大學
系所名稱(中) 細胞生物與解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 107
學期 2
出版年 108
研究生(中文) 林芷絹
研究生(英文) Chih-Chuan Lin
學號 T96064057
學位類別 碩士
語文別 英文
論文頁數 60頁
口試委員 指導教授-王仰高
口試委員-黃步敏
口試委員-邱文泰
中文關鍵字 微環境  肌腱分化  骨髓間質幹細胞 
英文關鍵字 Microenvironmental cues  tenogenesis  bone marrow-derived mesenchymal stem cell 
學科別分類
中文摘要 肌腱是一種結締組織,主要由平行膠原蛋白束組成。因為內在的癒合能力差,肌腱損傷涉及緩慢的修復過程。手術治療是肌腱斷裂目前最常見的治療方式。如今,研究人員致力於通過細胞療法研究肌腱修復,這被認為是一種理想的策略。 Scleraxis(Scx) 是一種轉錄因子,在肌腱/韌帶細胞群中表達,並在肌腱細胞中正向調節第一型膠原蛋(collagen I, Col1)和Tenomodulin (Tnmd) 的表達。先前的研究已經觀察到可溶性因子如結締組織生長因子(connective tissue growth factor, CTGF)和骨形態發生蛋白12 (bone morphogenetic protein 12, BMP12) 被證明參與了肌腱癒合過程的多個階段,增強了肌腱修復。另一方面,在物理環境中例如機械力,也顯示出能夠增強肌腱分化能力。雖然如此,物理力與生長因子的結合是否對肌腱再生有益卻很少受到關注。因此,本研究的目的是研究可溶性因子與物理因子的組合是否能夠加速幹細胞進行肌腱分化。我們在不同的時間和劑量下比較了BMP12 和CTGF。檢測肌腱分化相關標誌物Scx,Tnmd 和成骨標誌物Runx2 的蛋白質表達水平。結果表明,CTGF 對骨髓間質幹細胞的分化具有正面影響和特異性較高之特性。微環境控制細胞形狀,長寬比為1:1、3:1、7:1。免疫螢光檢測核中的Scx 表達,Tnmd 蛋白表達和統計核長寬比。結果發現,7:1 的長寬比可能是肌腱生成的適宜條件。此外,形狀控制與CTGF推進位於核中的Scx 相結合。利用細胞鬆弛素D抑制F-肌動蛋白聚合後,間接性破壞了形狀調控的影響,核中Scx 的表達也被抑制。在未來我們將確保細胞形狀調控的機制並結合CTGF 能夠加速肌腱生成。本研究可能有助於了解肌腱生成的確切條件,並進一步提供肌腱損傷的治療策略。
英文摘要 Tendon is a type of connective tissue which is mainly composed of parallel collagen bundles through the direction of mechanical load. Tendon injury involves a slow repair process, which results in the poor intrinsic healing capacity. Surgical treatment of tendon ruptures and lacerations is currently the most common therapeutic treatment. Nowadays, researcher devoted to studying the restoration of tendon rupture by cell therapy, which has been suggested to be an ideal strategy. Scleraxis (Scx), a basic helix-loop-helix transcription factor, is expressed in the tendon/ligament cell population and positively regulates the expression of type I collagen (Col1) and tenomodulin (Tnmd) in tenocytes. Previous study has observed soluble factors such as connective tissue growth factor (CTGF) and bone morphogenetic protein 12 (BMP12) are demonstrated to be participated in the multiple stages of tendon healing process enhancing tendon repair. On the other hand, physical cue, such as mechanical force, is shown to enhance the differentiation ability for tendon tissue engineering. Although several studies have indicated, little attention has been paid whether the combination of physical force with growth factors has beneficial effect on tendon regeneration. Thus, the objective of this study was to investigate whether the combination of soluble factors with physical factor accelerates tenogenesis. We compared BMP12 with CTGF in different times and dosages. Examine protein expression level of tenogeneic related marker Scx, Tnmd and osteogenic marker Runx2. Result showed CTGF possess a positive impact and specificity on MSC tenogenesis. Micropatterning control cell shape with the aspect ratio 1:1, 3:1, 7:1. Immunofluorescence detected Scx expression in nuclear, Tnmd protein expression and statistic nuclear aspect ratio. Result showed the aspect ratio 7:1 could be the proper condition for MSC tenogenesis. In addition, shape control combines with CTGF advancing Scx located in nuclear. After inhibited F-actin polymerization by Cytochalasin D, indirectivity destructs the shape control tenogenesis, the expression of Scx in nuclear also been inhibited. in the future we will make sure the mechanism of cell shape combines CTGF accelerate tenogenesis. The present study may help understand the precise condition for tenogenesis and further provide a therapeutic strategy for tendon injury.
論文目次 Abstract I
中文摘要 III
誌謝 IV
Chapter 1 Introduction 1
1-1 General Background Information 1
1-2 Tendon anatomy structure and tenocytes 1
1-3 Tendinopathy, healing process and cell therapy 2
1-4 Human mesenchymal stem cells 5
1-5 Tenogenesis mechanisms 6
1-6 Soluble factors regulate tenogenesis 6
1-7 Physical cues in tenogenesis 7
1-8 Extracellular matrix, cytoskeleton dynamic and integrin-mediated signaling 8
Hypothesis 11
Chapter 2 Materials and Methods 12
2-1 Experimental design 12
2-2 Cells and cell culture 12
2-3 MTT assay 13
2-4 Flow cytometric analysis 14
2-5 Osteogenic, adipogenic, and chondrogenic differentiation 15
2-6 Western blots 16
2-7 Immunofluorescent staining and quantification 16
2-8 Fabrication of micropatterned substrate 16
2-9 Colocalization analysis 18
2-10 Statistical analyses 18
Chapter 3 Results 19
3-1 Proliferation capacity of human bone marrow-derived mesenchymal stem cells (hMSCs) 19
3-2 Expression of surface markers of hMSC 19
3-3 Multilineage capacity of hMSC in vitro 19
3-4 CTGF induces hMSC tenogenesis better than BMP12 20
3-5 Elongated shape induces hMSC tenogenesis 20
3-6 Combination of CTGF and shape control accelerate tenogenesis 22
3-7 Cytoskeleton involved in shape control 23
Chapter 4 Discussion 25
Figures 28
Figure 1. Proliferation of human bone marrow-derived mesenchymal cells (hMSCs) 28
Figure 2. Flow cytometry analysis of the cell surface markers on hMSCs 29
Figure 3. Multi-differentiation potential of hMSCs 30
Figure 4. CTGF induces protein expression of tenogenic marker Scx and Tnmd but inhibits osteogenic marker Runx2 32
Figure 5. A schematic illustration of micropatterning and immunofluorescences of fibronectin coated condition 33
Figure 6. Elongated shape induces hMSCs tenogenesis 35
Figure 7. hMSCs grown on micropatterned surfaces with different aspect ratios 37
Figure 8. Combining soluble CTGF and shape control accelerate tenogenesis in day 1 and day 4 41
Figure 9. Establishment of CTGF and fibronectin coating on micropattern 42
Figure 10. Combine coating CTGF and shape control accelerate tenogenesis in day 1 and day 4 46
Figure 11. Nocodazole inhibit microtubule polymerization 47
Figure 12. Shape control-induced tenogenesis is microtubule independent 50
Figure 13. Combination of shape control and CTGF induced tenogenesis were actin dependent 54
References 55
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