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研究生:詹雅琪
研究生(外文):Ya-Chi Zhan
論文名稱:鯉魚魚皮之鋅及43kDa「鋅結合蛋白質」之分佈及其特性
論文名稱(外文):Distribution and characteristics of Zn and 43 kDa Zn-binding protein in the skin of common carp
指導教授:鄭森雄鄭森雄引用關係
指導教授(外文):Sen-Shyong Jeng
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:61
中文關鍵詞:鯉魚魚皮
外文關鍵詞:common carpfish skinZn
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摘要

鯉魚消化道組織高鋅現象來自於結合於其 fibroblast 上之
43 kDa 「鋅結合蛋白質」。鯉魚魚皮也含有甚多 fibroblast ,為知鯉魚魚皮之含鋅情形,及其與消化道組織之異同,乃進行本實驗。由實驗得知,鯉魚魚皮經乾式灰化後其總鋅濃度為 88±24 µg/g fresh tissue,其中有~90%之鋅可為 EDTA抽取。由此可知鯉魚魚皮中之鋅約有90%是鬆散地與其他物質結合,是可游離的鋅;只有~10%的鋅是與其他物質緊密地結合在一起。將魚皮分成背部、腹部及尾部,經次細胞分畫後,得知背、腹、尾部平均整體鋅濃度為85~119 µg/g fresh tissue ;其中以nuclei/cell debris fraction 中所占之鋅最高,佔整體魚皮的 82% 。草魚、鰱魚、吳郭魚之魚皮,整體鋅含量為 38~53 µg/g fresh tissue ,此三種魚魚皮之鋅含量約為鯉魚之 50%。另外,由實驗得知鯉魚魚皮 hydroxyproline 平均363 µg/g fresh tissue 比其他魚 (草魚、鰱魚、吳郭魚) 之平均 280±62 µg/g fresh tissue 含量較高。以含 4 M guanidine hydrocholoride 之lubrol 抽出鯉魚與其他魚 (草魚、鰱魚、吳郭魚) 魚皮之「鋅結合蛋白質」,發現鋅抽出率甚低。將同樣條件之抽出液與 43 kDa 「鋅結合蛋白質」抗體進行免疫化學分析後,結果發現,只有鯉魚魚皮抽出液,呈現免疫化學反應。另外,由免疫組織化學染色得知,只有鯉魚真皮層處,對 43 kDa 「鋅結合蛋白質」抗體呈現反應。草魚、鰱魚、吳郭魚真皮層,則幾無免疫反應。以上結果指出, 43 kDa 「鋅結合蛋白質」亦存在於鯉魚魚皮之真皮層中,極可能同樣係位於 fibroblast 上。
Abstract

Common carp always has high concentration in its digestive tract tissue, and it is known this high Zn exists in the plasma membranes of fibroblasts. The skin of the fish also riches in fibroblasts. Distribution of Zn in the skin of common carp was studied. It was found that the Zn concentration in the skin of common carp was 88 µg/(g fresh tissue) determined by dry-ashing method. About ~90% of the zinc in the skin of common carp could be extracted by EDTA solution, which means only ~10% of Zn was bound closely to some substance. The skin of the fish was divided into dorsum, abdomen and tail portion, After subcellular fractionation, it was found that in the dorsum, abdomen and tail, the average Zn were 85~119 µg/(g fresh tissue). About 82% of the Zn existed in the nuclei/cell debris fraction. The Zn concentration in skin of grass carp, silver carp and tilapia was 38~53 µg/(g fresh tissue), only about 50% of that in common carp. The hydroxyproline concentration in skin of common carp (363 µg/[g fresh tissue]) was higher than that in the other fish (281 µg/[g fresh tissue]). Zn-binding protein in the skin of common carp, grass carp, silver carp and tilapia was extracted by detergent lubrol with 4 M guanidine hydrochloride, and it was found that little Zn was extracted. Immunoassay of the guanidine/lubrol with the antibody against 43 kDa Zn-binding protein , indicated that only the extracts of common carp was positive. The immunohistochemical staining, indicated that the 43 kDa Zn-binding protein only exist in dermis of common carp. No immunoreaction was found in dermis of grass carp, silver carp and tilapia. The above mentioned results indicated that 43 kDa Zinc-binding protein also existed in dermis of common carp which most probably located in fibroblasts.
摘要-------------------------------------------------------------------------------- I
第一章研究背景及目的-----------------------------------------------------1
一、研究背景-------------------------------------------------------------------- 1
二、研究目的-------------------------------------------------------------------- 3
第二章文獻整理---------------------------------------------------------------4
一、魚類對鋅之需求------------------------------------------------------------4
二、魚類魚皮---------------------------------------------------------------------4
三、結締組織---------------------------------------------------------------------5
四、膠原蛋白---------------------------------------------------------------------6
五、collagen 與收縮溫度 (shrinkage temperature; Ts)
之關係--------------------------------------------------------------7
第三章材料與方法------------------------------------------------------------8
一、實驗用魚---------------------------------------------------------------------8
二、實驗儀器-------------------------------------------------------------------- 8
三、實驗方法--------------------------------------------------------------------9
(一)、鯉魚皮膚次細胞分劃------------------------------------------------9
(二)、化學分析---------------------------------------------------------------10
1、鋅的測定-----------------------------------------------------------------11
2、Hydroxyproline 之測定----------------------------------------------12
3、魚鱗之鋅測定------------------------------------------------------------12
(三)、以含 4 M guanidine hydrocholoride 之lubrol抽出鯉魚皮上之「鋅結合蛋白質」-------------------------------------12
(四)、以「鋅結合蛋白質」之 Antibody 進行 immunoassay---------12

(五)、組織切片製作---------------------------------------------------------13
1、固定------------------------------------------------------------------------13
2、脫水浸臘------------------------------------------------------------------14
3、包埋------------------------------------------------------------------------14
4、切片------------------------------------------------------------------------15
5、組織切片之 Elastin stain-------------------------------------------15
6、組織切片之免疫組織化學方法染色---------------------------------17
第四章 實驗結果------------------------------------------------------------20
一、鯉魚魚皮取樣--------------------------------------------------------------20
二、鯉魚魚皮中鋅之結合型態-----------------------------------------------20
三、鯉魚與其他魚魚皮 (草魚、鰱魚、吳郭魚) 之次細胞分畫-------21
四、鯉魚與其他魚魚皮 (草魚、鰱魚、吳郭魚) nuclei/cell debris fraction 與 collagen 之關係---------------------------------------22
五、以含 4 M guanidine hydrocholoride 之lubrol 抽出鯉魚與其他魚(草魚、鰱魚、吳郭魚) 魚皮之「鋅結合蛋白質」---------------22
六、 鯉魚及其他三種魚魚皮之 4 M guanidine hydrocholoride 之lubrol 抽出液對鯉魚 43 kDa 「鋅結合蛋白質」抗體之免疫化學反應----------------------------------------------------------------23
七、鯉魚與其他三種魚(草魚、鰱魚、吳郭魚)魚皮之組織化學觀察-23
(a) 鯉魚---------------------------------------------------------------------23
(1) Elastin stain-----------------------------------------------------------23
(2)以 43 kDa「鋅結合蛋白質」抗體進行組織免疫染色----------24
(b) 草魚、鰱魚、吳郭魚---------------------------------------------------24
(1) Elastin stain-----------------------------------------------------24
(2) 以 43 kDa「鋅結合蛋白質」抗體進行組織免疫染色----------24
第五章 綜合討論------------------------------------------------------------25
圖表-------------------------------------------------------------------------------27
參考文獻------------------------------------------------------------------------45
參考文獻

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