|Abstract: ||全文摘要 鯉魚消化道組織常具有高濃度之鋅（300-500μg/g fresh tissue），為其他水產生物之10-15倍。由次細胞分畫，已知鯉魚高濃度之鋅有約80%係在於其nuclei/cell debris fraction，而只有~20%存在於其cytosol、mitochondria /lysosome及microsome中。存在於nuclei/cell debris fraction中之鋅，係結合於膜蛋白質。為溶出鯉魚消化道組織nuclei/cell debris fraction中之「鋅結合蛋白質」，乃以9種清潔劑進行抽出實驗，結果發現以非離子性清潔劑n-Dodecyl-β-D-maltoside溶出「鋅結合蛋白質」效果最好。然而此清潔劑亦只能抽出~10﹪之「鋅結合蛋白質」。由此推論鯉魚消化道組織之「鋅結合蛋白質」，極可能係連接於胞外母質，或胞內之細胞骨架上，以致無法被清潔劑完全抽出。 在實驗中，本實驗室注意到鯉魚消化道組織比草魚、鰱魚與吳郭魚’’堅韌’’；為知此現象與其collagen含量之關係，乃分析此等魚類消化道組織之拉張力、collagen及鋅之含量，結果發現鯉魚消化道組織之鋅濃度大於其他魚10倍以上，拉張力與collagen含量分別為其他魚之 >5倍及2倍。鯉魚消化道組織鋅濃度較高時，拉張力即較高。另外，鋅濃 度與collagen含量亦成正比。將鯉魚、草魚、鰱魚及吳郭魚之消化道組織分別以 periodic acid Schiff stain及silver stain染色後，發現鯉魚之glycosaminoglycans甚為緻密且面積甚廣；與鯉魚同科之草魚與鰱魚，則較為狹窄稀少；吳郭魚之glycosaminoglycans所佔面積雖廣，但甚為稀鬆。由此可知，在鯉魚消化道組織鋅濃度遠大於草魚、鰱魚及吳郭魚之同時，其collagen及glycosaminoglycans之量亦明顯高出其他魚許多。 以載玻片將鯉魚消化道組織之黏膜刮除，分劃成”黏膜” （epithelial layer）及”去黏膜組織” （包含lamina propria、submucosal layer、muscular layerr及serosa）二部分；結果發現鯉魚消化道組織中有~76﹪之鋅及 ~74﹪之Bound SH group存在於”去黏膜組織”；即鯉魚消化道組織之「鋅結合蛋白質」主要存在於其”去黏膜組織”中。以鯉魚消化道組織之43 kDa「鋅結合蛋白質」抗體，進行免疫組織化學染色，並與Verhoeff elastin 染色比較；結果發現「鋅結合蛋白質」主要存在鯉魚消化道組織之lamina propria及submucosal layer，epithelial layer無免疫反應，muscular layer及serosa 則似有微弱反應。然而草魚、鰱魚及吳郭魚消化道組織切片， 對「鋅結合蛋白質」幾乎沒有免疫反應。以Thy 1.1 protein （fibroblasts 之marker）抗體對鯉魚消化道組織進行免疫組織化學染色，發現其具有免疫反應之區域，為lamina propria及submucosal layer，與「鋅結合蛋白質」免疫反應位置相同。 為了解「鋅結合蛋白質」存在於鯉魚消化道lamina propria及submucosal layer之何種細胞，乃將鯉魚消化道之樹脂切片以Giemsa染色。實驗結果發現lamina propria及submucosal layer之主要細胞，為直徑~6 μm之圓形細胞。免疫組織化學染色之結果，指出「鋅結合蛋白質」係位於此圓形細胞。由細胞之數量及其存在位置，此~6 μm之圓形細胞極可能為fibroblasts。綜合上述實驗結果，可知鯉魚消化道組織中之高鋅，係結合於43 kDa「鋅結合蛋白質」，而此蛋白質極可能係存在於其結締組織fibroblasts之細胞膜上。|
英文摘要 Summary Extraoridinary high concentration of Zn （300~500μg/g fresh tissue）are often found in the digestive tract of common carp. The Zn concentration in the digestive tract of common carp is always >10 times higher than other aquatic organisms. Subcellular fractionation of the digestive tract tissue showed that about 80% of the Zn was in the nuclei/cell debris fraction, and only ~20% was in all other fractions, including cytosol, mitochondria/ lysosome and microsome fractions. It was suggested that the Zn-binding substance in the nuclei/cell debris fraction was probably a membrane protein. In this study, 9 different kinds of detergents were used to solubilize the Zn-binding protein in the nuclei/cell debris fraction. It was found that n-Dodecyl-β-D-maltoside, a nonionic detergent, could extract more Zn-binding protein than other detergents. However, even the detergent could only release only ~10% of the Zn-binding protein. It is very probably that the Zn-binding protein is either connected to extracellular matrix or cytoskeleton. Hence, it can not be extracted only with detergent. In the experiment, it is noticed that the digestive tract of common carp was much tougher than those other species (include grass carp Ctenopharyngodon idellus, silver carp Hopophthalimichthys modlitrix and tilapia Oreochromis auerus). The toughness and concentrations of collagen and Zn in the digestive tract of common carp, and the three other fishes were compared. It was found that common carp had higher Zn (>10 times), toughness and collagen content (>5 times and ~2 times, respectively) than the other species. The experiments also indicated that there was a positive correlation between Zn concentration and toughness in different common carp; the higher the Zn concentration, the tougher the tissue. A positive correlation between the Zn and collagen content in the digestive tract tissue of common carp is also found. Besides collagen, the distribution of glycosaminoglycans in the digestive tracts of the four fish species were also studied histologically. In parallel to a much higher Zn concentration in common carp, the stains of periodic acid-Schiff and silver in the lamina propria and submucosal layer of common carp digestive tract are also much broader and denser than all the three other species of fishes. Mucosa (where epithelial layer existed) of the digestive tract of common carp was scrapped with a slide glass and the mucosa was removed. It was found that the mucosa (epithelial layer) contained only 14% of the Zn, while 86% of the Zn remained in the de-mucosa tissue (including lamina propria, submucosal layer, muscular layer and serosa). To localize the Zn-binding protein in the digestive tract of common carp, tissue sections of the fish were stained with Verhoeff elastin stain and compared with an indirect immunoperoxidase staining method. It was found that the Zn-binding protein was located in the lamina propria and submucosal layer of the digestive tract of the fish. This protein was not detectable, however, in the epithelial layer. Little staining was discernible in the muscular and serosal layers. Immunoperoxidase staining also indicated that there were little or no staining in the sections of digestive tracts of grass carp, silver carp or tilapia. Immunohistochemical staining of the Thy 1.1 protein, (a marker for fibroblasts in the digestive tract of common carp), indicates that its location is almost identical to the Zn-binding protein. In order to localize the cells with the Zn-binding protein, tissue sections of the digestive tract of common carp were stained with Giemsa method and compared with immunohistochemical method. Giemsa stain shows that in the lamina propria and submucosal layer of the tissue, the most abundant cells are round cells with a diameter of ~6 μm. Immunohistochemical results indicate that the Zn-binding protein is localized in the round cells. Judged from the amount and location of the cells, the predominant ~6um round cells are most probably fibroblasts. The high Zn in the digestive tract of common carp is bound by the 43 kDa Zn-binding protein which most probably located in the plasma membrane of the fibroblasts embeded in its connective tissue.