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  • 學位論文

由靈芝子實體經萃取後之廢渣所製成之薄膜對於天竺鼠之傷口及組織纖維 母細胞之影響

The effects of the membrane from Ganoderma fruiting on skin wound and fibroblast behaviors of Guinea-pig

指導教授 : 蘇慶華
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摘要


摘 要 以幾丁質 (chitin) 為皮膚損傷之覆被材料已有成功之案例,並且已有商 品上市 BESC( BESCHITIN W );其幾丁質之來源係以甲殼類如螃蟹之外 骨骼製成。本研究則以具有 X丁質幾丁質成份之靈芝子實體經萃取後之廢 渣進行研究。原料經熱水萃取後,其廢渣經磨碎 嵽H9後以95%酒精處理去 除脂溶物;以0.1N HCl常溫處理,經 1N NaOH 加熱水解後再以0.1% 葩熐 次氯酸鈉漂白、洗淨,最終可得約原料 50% 重量之純白色絲狀粉末。其 化學組成為60% 眶敹葡萄糖及40%乙醯葡萄糖氨(N-acetyl-glucosamine) 之共聚多糖体。此純白絲狀粉末經冷 嵹悌嵹挫磭嶆足隻h孔性膜,膜之形 狀及厚度可隨意調整。本研究以直徑7cm,厚度0.1~0.2mm 之多孔性膜 進行動物試驗。動物以體重 380~480 克之天竺鼠進行試驗,經麻醉後剃 毛於背 部雙側進行全皮膚 (Full thickness) 之切除,切除面積為 1.5 x 1.5 cm2。分別於一側覆 蓋靈芝多孔性膜,另一側則以紗布或 BESCHITIN W 覆蓋;然後將實驗動物飼養於27℃、相 對濕度70%之培養 箱中。其後每五天觀察傷口之復原情形,並以組織切片觀察組織增生及細 胞間之交互作用。其結果經 student T test 檢定後,顯示覆蓋靈芝薄膜 之傷口面積於第 10 天顯著小於紗布 (P<0.05),且與BESCHITIN W 無 顯著差異。接著本研究也探討實驗材質 對天竺鼠真皮中之纖維母細胞的 增生及位移之影響。細胞增殖方面,將(0.01%)實驗材質懸 浮於 DMEM 培養液中用以培養細胞並於第1,3,6,9,13天計算細胞數目。而細胞位移實 驗則分 為兩部份,其一是將長滿於細胞培養皿中之一半細胞刮除,再以 含有實驗材質之DMEM 培養 G液培養細胞,而後連續觀察五天。再者則是 將實驗材質懸浮於1% agarose 中,再將agarose 置於培養皿中待其凝固 後再將細胞置於中央之小孔(1 mm),而後觀察其爬行於 agarose 與 培 養皿之界面的情形。結果顯示靈芝子實體經萃取後之廢渣對於纖維母細胞 之增生及位移均 有顯著的促進效果。

並列摘要


Chitin was known to be a wound healing enhancer, and membrane made from crab shell has been commercialized as a dressing for skin defect. Since fungi with chitin component in their cell wall, it''s a charming idea to use fungal mycelia as a material for wound management. We used a new membrane, which was made from extracted waste of Ganoderma tsugae manufacturing, to evaluate the potential as a wound dressing. The residue of fruiting body of G. tsugae previously extracted with hot water was further trted with 95% EtOH, 0.1N HCl, 1N NaOH at 85℃, and 0.1% sodium hypochlorate to remove undesirable component, especially the protein and pigment. The final product was white powder, about half of the original material by weight, with filamentous structure in mycelial form under scanning electronic microscopy. Chemical analysis revealed that it was a copolymer of glucose ( 60% ) and N-acetyl-glucosamine ( 40% ). The white powder was then woven into thin, porous circular sheet of 7.0 cm in diameter and 0.1 ~ 0.mm in thickness by filtration and lyophylization. Female guinea pigs weighing 380 ~ 480 g were used. After anesthetized with ketamine and pentobarbital by intraperitoneal injection, dorsal skin hairs were removed with electric clipper. Two wounds as mirror-image were made on the back by dissecting 1.5 x 1.5 cm2 skin in full thickness. Ganoderma membrane was applied on one wound randomly and gauze or commercial chitin membrane was u sed on the other. The wound areas were measured and photographed at days 5, 10 , 15, 20 postoperation. Histological examinations were also performed to revea l the interaction of tissue and dressing. The Ganoderma wound areas were signi ficant smaller than gauze''s on day 10. There was no significant difference of wound size between chitin sheet from crab and Ganoderma. In the second part of the present investing, fibrobsts from dermis layer of guinea pigs were used t o examined the effect of proliferation and migration enhanced by Ganoderma res idue. Cell numbers of fibroblast culture in DMEM were counted on the days 1 , 3 , 6 , 9 and 13 with or without suspending dressing materials (0.01 w/v). Mig ration of fibroblast were measured by removing a part of the grown cells on DM EM plate and counting the cells migrated aross the margin of the cut for 5 day s. In addition, a small hole (1 mm in diameter) was punched at the center aga rose (1%) plate suspended with dressing materials and 1-1.5×103 fibroblasts w ere put into the hole, then DMEM was flooded over agarose surface. Microscopic observation lasted for 5 days on the migration of fibroblast and the number c ell moved out of the hole were counted. All the results indicated that the Gan oderma suspension enhanced proliferation and migration of fibroblast, signific antly.

並列關鍵字

Ganoderma Guinea-pig fibroblast

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