臺灣博碩士論文加值系統

聚羥基脂肪酸酯(polyhydroxyalkanoates，PHA)，是由微生物細胞體內所大量儲藏當作能源的生物聚脂，目前已經發現其擁有近100多種單體架構，是幾十年間非常熱門且具有發展性的生醫材料，其擁有良好的生物相容性、生物可降解性、可熱加工性、氣體阻隔性、以及類似合成塑料的熱塑性和彈性等優秀的性質，使其做為生醫材料以及近期非常熱門的生物可分解的包裝材料都極其適合，而被大量用於生醫材料以及生物材料發展性的，主要為聚羥基脂肪酸酯中的聚羥基丁酸酯(polyhydroxybutyrate，PHB）和3-羥基丁酸酯和3-羥基戊酸酯的共聚物[poly(3-hydroxybutyrate-co-3-hydroxyvalerate)，PHBV]，但其缺點是機械性能差及與細胞結合率薄弱，也是目前科學家們想發展聚羥基脂肪酸酯及其聚合物所面臨的最大課題。
在本研究中，把甘油作為碳源，加入丙酸鈉前體，來培養來自活性污泥中的特定菌株Ralstonia eutropha H16使其在體內形成3-羥基丁酸酯和3-羥基戊酸酯的共聚物，通過溶劑鑄造製備的3-羥基丁酸酯和3-羥基戊酸酯的共聚物膜用等離子體(plasma-modified)進行表面改質，近而增加其生物相容性以及細胞貼附率並且改善機械性質不足的問題，並在材料上培養幹細胞，確認其是否能夠做為傷口修復相關的敷料，之後利用傅里葉變換紅外光譜(fourier transform infrared spectrometer，FTIR)和核磁共振光譜學(nuclear magnetic resonance spectroscopy， NMR)分析顯示，發現有官能基的增加，後對改質前後的PHB和PHBV進行表面的水接觸角的角度分析；幹細胞是原始且未特化的細胞其具有容易分化、再生各種組織器官潛在功能的一類細胞，而我們選用的是脂肪幹細胞(adipose-derived stem cells，ASCs)，經由MTT assay 測得在聚羥基脂肪酸酯中的聚羥基丁酸酯與3-羥基丁酸酯和3-羥基戊酸酯的共聚物改質前後的細胞生長率比較，並且用酵素免疫分析法(vnzyme-linked immunosorbent assay，ELISA)初步測得血管內皮生長因子(vascular endothelial growth factor，VEGF)和轉化生長因子-β (transforming growth factor-β，TGF-β)的分泌量，最後使用西方墨點法（western blot）和即時聚合酶鏈鎖反應（real-time polymerase chain reaction，全稱quantitative real time polymerase chain reaction，故簡稱qPCR/qRT-PCR）, 進而測得與血管內皮生長因子和轉化生長因子-β相關且與傷口修復有關連的蛋白質，驗証本研究的理論。經過種種的驗証及實驗，確實證明經由電漿法進行表面改質的聚羥基脂肪酸酯中的聚羥基丁酸酯和3-羥基丁酸酯和3-羥基戊酸酯的共聚物在生物相容性和機械性質上有相當的增強，並且脂肪幹細胞培養在材料上都有相當好的細胞貼附率，血管內皮生長因子和轉化生長因子-β分泌量測試有明顯增加，ERK and MEK, 也有相對應的表現量；經過等離子體表面改質後的生醫材料能夠改善其生物相容性和機械性質，使聚羥基脂肪酸酯中的聚羥基丁酸酯和3-羥基丁酸酯和3-羥基戊酸酯的共聚物更容易讓脂肪幹細胞增生以及使生長因子分泌更多，進而幫助傷口修復，運用於傷口修復的敷料上。

Polyhydroxyalkanoates (PHA) is a large bioenergy of storage as biopolyester in microbial cells. It has been found that it has nearly 100 kinds of monomer structure, which is very popular to develop biomaterials. It has excellent biocompatibility, biodegradability, hot workability, gas barrier properties, and similarity of synthetic plastics thermoplastic and elasticity and other excellent advantage. This are extremely as biomaterial and very popular of biodegradability packaging materials suitable, and was used for biomaterials and biological materials development. polyhydroxybutyrate (PHB) is one of the PHA and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). PHB are widely used in the development of biodegradability materials and biomaterials, but its shortcomings are significantly obvious: poor mechanical properties and weak cell attachment, There disadvantages are the reason for the current scientists to develop PHA, and its polymer is facing the biggest issue.
In this study, glycerol was added as a carbon source to the sodium propionate precursor for the production of PHBV from the designated strain ralstoniaeutropha H16 in activated sludge, and the PHBV biofilm who is prepared by solvent casting was subjected to plasma-modified surface modification. In addition to increasing its biocompatibility and cell attachment and improving the mechanical properties, it culture on stem cells to confirm whether it can be used as wound repair in the material. The results of Fourier Transform Infrared Spectrometer (FTIR) and nuclear magnetic resonance spectroscopy (NMR) showed that the increase of functional groups was found, the angle of water contacted the angle of PHB and PHBV before and after surface modification was analyzed；Stem cells are primitive and undifferentiation cells with a class of cells that are susceptible to differentiate and regenerate the potential function of various tissues and organs, and we use adipose-derived stem cells (ASCs) determination by MTT assay in PHB and PHBV before and after surface modification in cell growth rate. The levels of vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β) were determinate by enzyme-linked immunosorbent assay (ELISA). Finally, we selected some proteins interrelated with TGF-β and VEGF of wound repair that were obtained by western blot and real-time polymerase chain reaction (quantitative real time polymerase chain reaction, qPCR/qRT-PCR.
Based on the results, it is confirmed that PHB and PHBV were successful surface modified by plasma, and it have a considerable enhancement in biocompatibility and mechanical properties, and ADSC culture in the material have very well cell attachment, in the initial TGF-β and VEGF secretion test also significantly increased, further analysis of the proteins interrelated with wound repair of the wound has also very fruitful results, so the material after plasma modification can effectively make cell proliferation and angiogenic growth factors secretion increase which is more suitable for wound repair scaffold.

中文摘要
ABSTRACT
目錄
表目錄
圖目錄
Introduction
Biomaterials
Polyhydroxyalkanoates (PHA)
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) PHBV and polyhydroxybutyrate (PHB)
Surface modification
Adipose-derived stem cells
Materials and Methods
Preparation of films - Solvent casting of film
Plasma modification treatment
NMR
FTIR
Surface morphology
Water contact angle
Cell viability of ASCs culture in PHBV and PHB
Experiment of XTT assay to angiogenic growth factors by ASCs culture in PHBV and PHB.
Western blot analysis
Results
NMR Spectral Analysis
FTIR Spectral Analysis
Surface morphology
Contact angle measurements
Cell viability of ASCs culture in PHBV and PHB
Experiment of XTT assay to angiogenic growth factors by ASCs culture in PHBV and PHB.
The protein expression level
Discussion
Conclusion
Reference

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