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研究生:黃耀正
研究生(外文):Yao-Zheng Huang
論文名稱:利用匍枝根黴菌(Rhizopusstolonifer)細胞壁組成RHIZOCHITIN作為生物支架探討生物降解性及生物相容性
論文名稱(外文):Biodegradability and Biocompatibility of the Scaffold Derived from Cell Wall Component of Rhizopus stolonifer
指導教授:蘇慶華蘇慶華引用關係
指導教授(外文):Ching-Hua Su
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:75
中文關鍵詞:溶菌酶生物降解性生物相容性匍枝根黴菌
外文關鍵詞:LysozymeBiocompatibilityBiodegradabilityRhizopus stolonifer
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本研究利用延緩孢子產生的突變Rhizopus stolonifer F6菌株,經液體培養基培養得到海綿狀的菌絲墊。此菌絲利用1N NaOH 在121oC處理20分鐘後凍乾得到的物質稱為RHIZOCHITIN (RC)。RC再經95%酒精沖洗,以去除非皂化的酯類後得到的物質稱為alcohol-treated RHIZOCHITIN (ARC)。市售的正向控制組BESCHITIN-W (BC)與RC和ARC作為生物支架分別進行體外和體內的生物相容性和生物降解性測試。體外測試包括溶菌酶分解和纖維母細胞(L-929)培養,分別看生物支架前後重量改變與顯微鏡觀察(光學顯微鏡與電子顯微鏡)結構前後是否不同,得知溶菌酶在前三週RC和ARC材料重量減少明顯比BC還要少,表示RC和ARC不會很明顯的被溶菌酶分解,反之BC就明顯的被分解,在SEM中也可看到三者結構的改變。RC和ARC不會影響纖維母細胞的存活,並且在細胞培養中也可以發現,RC和ARC材料重量減少,可知L-929會對RC和ARC產生分解作用,但是BC不僅會降低L-929存活率外,而且只可以分解少許的材料。體內研究證明將RC和ARC植入SD大鼠皮下組織發現在兩週已完全被分解,然而BC在四週時還可被取出,表示BC在體內分解速度慢於RC和ARC。實驗中RC、ARC和BC植入皮下組織均不會造成皮膚過敏和刺激性現象產生。最後,在植入後四週,利用直徑為8 mm大小的鑽孔器,在植入的區域取出組織,進行HE和PAS染色,兩者結果均顯示RC和ARC在體內被分解的速度遠大於BC。本研究得知從R.stolonifer F6細胞壁組成成分的物質,是具有生物相容性與生物降解性。
In the present study, the sporangia-formation-delayed mutant strain Rhizopus stolonifer F6 was used to obtain mycelium mattress by liquid culture. The mycelium mattress was treated with 1N NaOH at 121oC for 20 minutes and followed by lyophilization to have RHIZOCHITIN (RC). RC was then again washed with 95% alcohol to remove non-saponification lipid to get alcohol-treated RHIZOCHITIN (ARC). RC and ARC together with a commercial product BESCHITIN-W (BC) for positive control were employed as scaffold for the tests of biocompatibility and biodegradability in vitro and in vivo. In vitro tests included lysozyme degradation and co-culture to fibroblast cell line (L-929) with the weight change and microscopy (light and scanning electron microscopy). Lysozyme did not digest RC and ARC at the first three weeks but significant degradation was observed in the group of BC by the decrease in weight and the observation under SEM. RC and ARC did not affect the survival of fibroblast cell line in culture condition with an obvious decrease in weight. However BC significantly decreased the survival rate of the fibroblast cell with only slightly degradation of the scaffold. In vivo study demonstrated that RC and ARC had totally degraded after 2 weeks imbedded in subcutaneous layer of SD rats and BC showed a pattern of slow degradation through a four-week observation. No allergic reaction or irritation on the skin of the rats was observed through the experiment for RC ARC and BC. Finally, the tissues of the embedded area were sampled by a punch of 8 mm in diameter after 4 weeks after imbedding of the scaffolds and section of the tissues revealed that RC and ARC were far more degradable than BC in both HE and PAS stains. The study suggested that the cell wall component derived from Rhizopus stolonifer F6 was biocompatible and biodegradable.
中文摘要………………………………………………………………………………1
英文摘要………………………………………………………………………………2
第一章 緒論…………………………………………………………………………..4
1-1、細胞(Cell) ……………………………………………………………………4
1-2、訊號(Signal) ………………………………………………………………….5
1-3、生物支架(Scaffold)…………………………………………………………..5
1-3-1、生物相容性(Biocompatibility)…………………………………..………6
1-3-2、生物降解性(Biodegradation)……………………………………………..7
1-3-3、溶菌酶 (Lysozyme)....................................................................................7
1-4、天然材料……………………………………………………………………..8
1-4-1、明膠(Gelatin)…………………………………………………….……….8
1-4-2、膠原蛋白(Collgen)……………………..…………………………………8
1-4-3、玻尿酸(Hyaluronic acid)…………………………………………………9
1-4-4、幾丁質及幾丁聚醣(Chitin & Chitosan)………………………………10
1-5、本實驗先前研究……………………..…………………………………….14
1-5-1、理想敷料之特性......................................................................................14
1-5-2、真菌性幾丁質及幾丁聚糖.................................................................... .15
1-5-3、Rhizochitin And Rhizochitosan................................................................16
第二章 研究動機........................................................................................................18
第三章 研究架構........................................................................................................19
第四章 研究方法與材料............................................................................................21
4-1、研究材料........................................................................................................21
4-2、研究方法........................................................................................................23
4-2-1、敷料的製備流程.......................................................................................23
4-2-2、材料吸水性測定...................................................................................25
4-2-3、材料外觀觀察........................................................................................25
4-2-4、材料生物相容性....................................................................................26
4-2-5、材料生物降解性....................................................................................28
第五章 實驗結果........................................................................................................33
5-1、材料的製備....................................................................................................33
5-2、材料吸水性測試............................................................................................34
5-3、材料生物相容性測試....................................................................................34
5-4、材料生物降解性測試....................................................................................35
第六章 討論................................................................................................................39
6-1、RHIZOCHITIN作為材料來源原因..............................................................39
6-2、材料的成分與溶菌酶作用相關性.................................................................40
6-3、細胞毒性與細胞影響生物支架降解關係.....................................................41
6-4、材料的成分與細胞作用相關性.....................................................................42
6-5、材料吸水性測試.............................................................................................43
6-6、材料的成分在動物體影響.............................................................................43
6-7、材料的成分在動物體中降解的應用.............................................................45
第七章 結論................................................................................................................48
第八章 參考文獻........................................................................................................49
論文圖表......................................................................................................................55
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