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

1. 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 • 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 1.5x 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。

2. The effects of the membrane from Ganoderma fruiting on skin wound and fibroblast behaviors of Guinea-pig • Chitin was known to be a wound healing enhancer, andmembrane made from crab shell has been commercialized as adressing for skin defect. Since fungi with chitin component intheir cell wall, it&apos;&apos;s a charming idea to use fungal mycelia as amaterial for wound management. We used a new membrane, which wasmade from extracted waste of Ganoderma tsugae manufacturing, toevaluate the potential as a wound dressing. The residue offruiting body of G. tsugae previously extracted with hot waterwas further trted with 95% EtOH, 0.1N HCl, 1N NaOH at 85℃, and0.1% sodium hypochlorate to remove undesirable component,especially the protein and pigment. The final product was whitepowder, about half of the original material by weight, withfilamentous structure in mycelial form under scanning electronicmicroscopy. Chemical analysis revealed that it was a copolymerof glucose ( 60% ) and N-acetyl-glucosamine ( 40% ). The whitepowder was then woven into thin, porous circular sheet of 7.0 cmin diameter and 0.1 ~ 0.mm in thickness by filtration andlyophylization. Female guinea pigs weighing 380 ~ 480 g wereused. After anesthetized with ketamine and pentobarbital byintraperitoneal injection, dorsal skin hairs were removed withelectric clipper. Two wounds as mirror-image were made on theback by dissecting 1.5 x 1.5 cm2 skin in full thickness.Ganoderma membrane was appliedon one wound randomly and gauze or commercial chitin membranewas u sed on the other. The wound areas were measured andphotographed at days 5, 10 , 15, 20 postoperation.Histological examinations were also performed to revea l theinteraction of tissue and dressing. The Ganoderma wound areaswere signi ficant smaller than gauze&apos;&apos;s on day 10. There was nosignificant difference of wound size between chitin sheetfrom crab and Ganoderma. In the second part of the presentinvesting, fibrobsts from dermis layer of guinea pigs were usedt o examined the effect of proliferation and migrationenhanced by Ganoderma res idue. Cell numbers of fibroblastculture in DMEM were counted on the days 1 , 3 , 6 , 9 and 13with or without suspending dressing materials (0.01 w/v). Migration of fibroblast were measured by removing a part of thegrown cells on DM EM plate and counting the cells migratedaross the margin of the cut for 5 day s. In addition, a smallhole (1 mm in diameter) was punched at the center aga rose(1%) plate suspended with dressing materials and 1-1.5×103fibroblasts w ere put into the hole, then DMEM was floodedover agarose surface. Microscopic observation lasted for 5days on the migration of fibroblast and the number c ell movedout of the hole were counted. All the results indicated that theGan oderma suspension enhanced proliferation and migration offibroblast, signific antly.