臺灣博碩士論文加值系統

摘 要
近年來的研究發現，蓴菜含有豐富的多醣及植物性蛋白等營養成分，蓴菜的食用部分即為富含膠質的嫩梢，包括嫩芽及未展開的幼葉；蓴菜的產地包括台灣宜蘭縣、中國大陸西湖杭州一帶及美國德州、日本、韓國、澳洲等地（Osborn and Schneider, 1988） （Lloyd and Kershaw, 2004）。許多研究顯示，蓴菜富含多醣、植物性蛋白及鐵、鈣、鎂、鋅、鉀、錳、磷等礦物質和Vit. C、Vit.B1、B2、B6、B12以及人體必須的八種氨基酸。如果能善加利用蓴菜多醣中特有的組成成份，相信將有助於多醣類運用的多重選擇性。近來生物科技蓬勃發展，帶動了相關生醫科技的研發與應用；其中生長分化因子具誘導及調控的角色，對細胞分化及間質的分泌都有決定性的影響。而許多很多研究發現血小板除了止血效用外，尚有釋放生長因子修復組織及調節體內代謝等許多功能，故生物醫學界極思利用血小板內蘊含的大量生長分化因子應用於引導組織修護及再生。
本實驗即期望利用多醣類聚電解質複合物(Polyelectrolyte)之特性，藉由蓴菜多醣溶液將血小板有效的吸附至環狀糊精上，使血小板於體外受到良好的包被保護，以利各種生醫及美容保養方面等應用。首先以酒精沈澱法製備蓴菜多醣並凍乾保存後；再以生理食鹽水復水稀釋，應用於血小板體外保存包覆。再以生長因子套件測試經包被後凍乾完成之血小板顆粒，討論其生長因子釋放情形，來推論蓴菜多醣應用於血小板體外保存之效用。本實驗之主要目的，即希望由蓴菜萃取之多醣可有效保護血小板，並可將保護好之血小板吸附於生醫載體上。
經過第0、7、14、21、28及56天的生長因子釋放發現，以三種pH值蓴菜多醣溶液來包被之血小板環狀糊精吸附物，其生長因子釋放情形以pH 6蓴菜組較佳，在EGF、TGF及PDGF等生長因子的釋放濃度均較其他兩組高出許多。而以蓴菜多醣、幾丁聚醣、膠原蛋白及羧甲基纖維素溶液包被之血小板環狀糊精吸附物，其生長因子釋放情形仍以蓴菜組較佳。

Abstract
Junsai (Brasenia schreberi J.F. Gmel), a water plant belongs to the lotus family, grows in swampy areas in Taiwan, China, Japan, korea, Australia or USA etc..(Osborn and Schneider, 1988; Lloyd and Kershaw, 2004). Junsai has been reported that it contains rich of polysaccharides , proteins, minerals such as Fe, Ca, Me, Zn, Ka, Mn and P, some water soluble vitamins such as Vitamins C, B1, B2. B6, and B12 and some human essential amino acids. Traditional, it has been used as a vegetable in Japan, China etc.. The edible parts are the young leave and stem that usually coated with a transparent mucilages. The mucilage is very rich in polysaccharides. In the last ten years, the study and research developments of the application of polysaccharides in biotechnology expanded rapidly. If the usage of Junsai polysaccharides could be extended to new areas such as cosmetics or biomedical materials, this will elevate the value of Junsai and provide the chosen alternates of variety polysaccharides. Increasing evidences showed that platelets are not more than the plug to stop bleeding, but also has the ability to release a variety of growth factors that can modulate tissue repaired and metabolism regulation. The goal of this study is to explore the capacity of protective effect of the polysaccharides of Junsai on the platelets-cyclodextrins attached complex by ionic interactions to extend the storage life of platelet to be used in various biomedical applications.
The polysaccharides of Junsai was separated by ethanol, and frozen dried at -30�aC. The freezing dried powder was dissolved in an isotonic sodium chloride solution of three different pHs. The solutions were used to coat the platelets-cyclodextrins attached complex by the counter-ion effect of the polyelectrolytes of Junsai polysaccharides. The release of growth factors of EGF, TGF and PDGF were measure by the respectively ELISA kits to compare and elucidate the protection effect of different pH polysaccharide solutions of Junsai. The protective effect of the solutions of chitosan, collagen or sodium carboxy methyl cellulose were also studied.
The data of the activities of growth factors release from the platelets-cyclodextrins attached complex coated with pH 6 polysaccharides solution of Junsai could be retained higher amount and longer periods than those coated with other two pHs solution of 5 or 4 on the zero, 7th, 14th, 21st, 28th and 56th days. The platelets-cyclodextrins attached complex coated with pH 6 polysaccharides solution of Junsai showed better protection effect than those coated with chitosan, collagen or Sodium carboxy methyl cellulose solutions.

目 錄
中文摘要………………………………………………………………Π
英文摘要………………………………………………………………Ⅳ
目錄……………………………………………………………………Ⅵ
一、前言………………………………………………………………1
二、文獻整理…………………………………………………………1
1.蓴菜的簡介…………………………………………………………1
2.蓴菜多醣的應用……………………………………………………3
3.聚電解質複合物……………………………………………………3
4.血小板(Platelet)及生長因子(Growth factors)的簡介………5
5.血小板的儲藏………………………………………………………6
三、材料與方法………………………………………………………7
1.實驗大綱……………………………………………………………7
2.實驗原料、藥品與儀器設備………………………………………8
2.1樣品………………………………………………………………8
2.2標準品……………………………………………………………8
2.3藥品與其他材料…………………………………………………8
2.4儀器設備…………………………………………………………9
2.5幾丁聚醣的製備…………………………………………………9
3.實驗方法……………………………………………………………11
3.1蓴菜抽出物的製備………………………………………………11
3.2蓴菜多醣的沈澱劃分……………………………………………12
3.3蓴菜一般組成份測定……………………………………………12
3.4蓴菜多醣包覆血小板(Platelet)之應用
-血小板環狀糊精顆粒(PCAC particle)的製備………14
3.5血小板體外保存之有效性測試…………………………………15
3.6測試生長因子之釋放量…………………………………………16
3.6-1TGF-β1…………………………………………………………16
3.6-2 PDGF-AB…………………………………………………………17
3.6-3 EGF………………………………………………………………18
3.7.生長因子控制釋放及保存活性試驗……………………………19
3.8.統計分析…………………………………………………………20
四、結果與討論………………………………………………………20
1.蓴菜多醣的萃取率及一般組成分析………………………………20
2. 血小板體外保護之有效性………………………………………20
3.不同pH值之蓴菜多醣作為血小板環狀糊精吸附顆粒之應用結果
…………………………………………………………………………21
3.1 EGF………………………………………………………………21
3.2.TGF………………………………………………………………21
3.3.PDGF………………………………………………………………22
4.推測不同pH值及醋酸保存溶液對蓴菜多醣的降解影響………22
5.不同生物聚合物對血小板保護情形之比較………………………23
5.1.EGF………………………………………………………………23
5.2.TGF………………………………………………………………24
5.3.PDGF………………………………………………………………24
6.不同生物聚合物對血小板保護之可能影響因素………………25
6.1.電荷效應（聚電解質）的影響…………………………………25
6.2.黏度的影響………………………………………………………25
五、結論………………………………………………………………27
六、參考文獻…………………………………………………………28
七、圖表………………………………………………………………Ⅸ

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