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系統識別號 U0026-2402201509190700
論文名稱(中文) 不同頻率之往復雙軸拉伸對於含纖維母細胞之膠原蛋白凝膠的影響
論文名稱(英文) Effects of the frequency of cyclic biaxial stretching on fibroblast-seeded collagen gels
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 103
學期 2
出版年 104
研究生(中文) 簡妙兒
研究生(英文) Miao-Erh Chien
學號 P86014218
學位類別 碩士
語文別 中文
論文頁數 50頁
口試委員 指導教授-胡晉嘉
口試委員-黃玲惠
口試委員-吳梨華
口試委員-邱文泰
口試委員-王仰高
中文關鍵字 含細胞之膠原蛋白凝膠  動態力學刺激  拉伸頻率  肌纖維母細胞 
英文關鍵字 fibroblast-seeded collagen gels  α-SMA  stretching frequency  myofibroblasts 
學科別分類
中文摘要 機械性質的不足在天然聚合物所製備的人工組織的一大問題,在先前的研究當中機械力的刺激已經被證實在提高人工組織的機械性質與促進胞外基質的重塑上扮演重要的作用。本研究使用含細胞之膠原蛋白凝膠為模型,將之培養在頻率不同之動態力學刺激當中,這包括了定應變之等雙軸往復式拉伸 (CES-7%; CES-20%)以及動態單軸往復式拉伸(CSBS),自由懸浮培養與等雙軸固定拉伸則作為對照組。在六天的培養後,我們以非線性光學顯微鏡之二倍頻與雙光子吸收分別觀察膠原蛋白纖維及細胞,以組織染色觀察其細胞並且使用H&E染色影像來分析細胞的排列分布程度,也以免疫組織化學染色觀察其細胞增生、凋亡以及平滑肌動蛋白的表現,及以Western-Blot定量平滑肌動蛋白表現。我們發現,高頻率(1 Hz)的動態拉伸與低頻率(0.4 Hz)的動態拉伸對於細胞的排列程度上沒有顯著的差異,在受雙軸刺激的區域中,細胞的排列程度高低為CSBS>CES-20%>CES-7%;此外在受單軸刺激的區域當中,細胞的排列程度高低為CES-20%>CSBS>CES-7%。並且我們在非線性光學顯微鏡之二倍頻與雙光子吸收觀察膠原蛋白纖維的排列也發現,受到單軸刺激的區域膠原蛋白凝膠纖維會明顯朝著受拉伸的軸平行排列。於細胞的型態和分化上,動態拉伸(CES-7% ; CES-20%和CSBS)細胞增生的表現量較等雙軸固定拉伸培養來的不明顯,Apoptosis則在自由懸浮凝膠內的細胞有高度表現,在動態力學拉伸的情況下則較無表現。而於α-SMA的表現則為CES-20%高度表現,而等雙軸固定拉伸則無明顯表現,但於較小應變CES-7%和CSBS之動態力學培養則又再次表現α-SMA。綜合以上,動態刺激的頻率高低對於含細胞之膠原蛋白凝膠之胞外間質的微結構與細胞的表現型有關,而動態力學刺激的培養對於組織的發展與條細胞型態表現較為劇烈。
英文摘要 Insufficient mechanical strength is a main problem of tissue engineered grafts fabricated from natural polymers. Mechanical stimuli have been proven as an important role in turnover of artificial tissues and matrix reorganization. In this study, we used fibroblast-seeded collagen gels as a model to study the mechono-biological responses under defined cyclic biaxial mechanical stretching. The fibroblast-seeded collagen gels were cultured under five different mechanical environments: free-floating, static equibiaxial stretching, constant strain amplitude cyclic equibiaxial stretching (CES-7% and CES-20%)and cyclic strip-biaxial stretching (CSBS). We used nonlinear optical microscopy to observe the collagen fibers and cell in the gel. Cell proliferation, apoptosis and expression of smooth muscle α actin were quantified by immunohistochemistry. Furthermore, western blot was used to quantify gene expression of collagen α-SMA. Results showed that the cell alignment were independent on the frequency of mechanical stretch. In the biaxial region of dynamic cultured environments cell alignment was CSBS>CES-20%>CES-7%, on the other hand in the uniaxial region of dynamic cultured environments cell alignment was CES-20%>CSBS>CES-7%. The image from nonlinear optical microscope illustrate that the gels cultured under dynamic cultured environments had the most collagen fiber alignment. No significance difference was observed in cell proliferation among gels cultured under dynamic cultured environments, but in the free-floating cultured condition cells would tend to apoptosis, while the expression of α-SMA showed opposite trends. In summary, the high-frequency of dynamic culture condition appeared to correlate the microstructure of the gels as well as the phenotype of the cells. Gels cultured under CES-20% regulate phenotype of fibroblast, remodeling of matrix, and turnover of cell.
論文目次 中文摘要 II
ABSTRACT III
致謝 VIII
目錄 IX
表目錄 XI
圖目錄 XII
1.1前言 1
1.2含纖維母細胞之膠原蛋白凝膠系統 2
1.3生物反應器 3
1.4力學刺激 4
1.5研究動機與目的 5
2. MATERIALS AND METHODS 6
2.1 實驗架構圖 6
2.2製備含細胞之膠原蛋白凝膠 7
2.3生物反應器與雙軸力學刺激環境 8
2.4 Hematoxylin and eosin stain (H&E stain) 9
2.5 Angular distribution of cell orientations 9
2.6免疫組織化學染色(Immunohistochemistry, IHC) 10
2.7 TUNEL檢測 10
2.8 非線性光學顯微鏡(Nonlinear optical microscopy) 10
2.9西方墨點法(Western blot) 11
2.10酵素免疫分析法(Enzyme-linked immunosorbent assay, ELISA) 11
2.11統計方法 11
3. RESULTS 12
3.1組織切片染色 12
3.2免疫組織化學染色細胞增生的表現 20
3.3 TUNEL細胞凋亡的表現 23
3.4免疫組織化學肌纖維母細胞分化之表現 26
3.5 ELISA-TGF-β濃度 30
3.6 Western Blot 32
4. DISCUSSION 34
5. CONCLUSION 38
6. REFERENCES 39
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