吳郭魚鰓 MR 細胞之研究：形態及運輸蛋白之表現

Abstract

MR 細胞（ mitochondria-rich cell ）為鰓上皮細胞中，主要負責離子、滲透壓調節的細胞。在淡水環境中， MR 細胞負責鈉、氯等離子的吸收。依掃瞄式電子顯微鏡觀察其頂部表面的結構，可將 MR 細胞區分成三型：突起型（wavy-convex）、淺盆型（shallow-basin）及深洞型（Deep-hole）。本實驗為了更進一步確認三型細胞在淡水環境下的角色，首先以缺氯水（F-Cl）、高鈉水（H-Na）及高氯化鈉水（H-NaCl），確認鈉、氯離子與三型MR細胞的關係。結果顯示，在缺乏氯離子的環境中（F-Cl）主要為突起型MR細胞，若在缺乏氯離子的環境中，外加 10mM 的鈉離子時（H-Na）,出現之MR細胞，仍以突起型細胞為主；另外，在環境中外加 10mM 的氯化鈉離子時（ H -NaCl），則突起型MR細胞消失，深洞型MR細胞產生。故突起型細胞的產生與環境中缺乏氯離子有關而與環境中的鈉離子無關。因此推論，突起型 MR 細胞為淡水環境中主要負責氯離子吸收的細胞。此外，缺氯環境下，鰓薄板上發現的 MR 細胞，還有大量表現的多細胞組合體，增加 MR 細胞頂部表面與外界接觸的面積，加強對外界環境離子吸收的能力。進一步以植物性凝集素 ConcanavalinA（ConA）配合 Na+,K+-ATPase做雙重染色，確定 Con A 確實染在 MR 細胞頂部表面的結構上，依 Con A 的染色特性將 MR 細胞區分成突起型（wavy-convex MR cell）及非突起型（non wavy-convex MR cell）。且兩型 MR 細胞皆有Na+,K+- ATPase 的表現，皆為淡水環境下，負責滲透壓離子調節之細胞。計算兩型 MR 細胞在 F-C1 、H-Na 和 H-NaCl 中的數量表現，結果發現，F-Cl、H-Na 中，主要表現突起型細胞；在 H-NaCl 主要表現非突起型細胞，此結果與 SEM 下所觀察到的結果相似，由此可知，Con A 為標定 MR 細胞極佳的標誌。Na+ , K+-ATPase西方墨點法的實驗證實，三種低張環境下，吳郭魚鰓上皆有Na+,K+-ATPase的表現，其中又以缺氯環境下（F-Cl 和H-Na）Na+,K+-ATPase的表現量最多。此意味著 MR 細胞為了調節對不同環境中各離子的吸收，進而調整Na+,K+-ATPase的表現，以利於 MR 細胞對不同離子調節之功能。

In gill epithelium, mitochondria-rich cells (MR cells) are considered to be the major cell type for ion-and osmo-regulation. In fresh water, gill MR cells are suggested to be responsible for ion uptake. Based on SEM (scanning electron microscope) observations on the apical surface, MR cells are classified into three types, wavy-convex, shallow-basin, and deep-hole types. Fish were acclimated to three hypotonic medium, F-Cl (chloride-free) ，H-Na (high Na2SO4) and H-NaCl (high NaCl), and the relative densities of the three types of MR cells were examined. The major cell type in chloride-free medium (F-Cl and H-Na) was wavy-convex, while that in H-NaCl was deep-hole. The major function of wavy-convex MR cells was suggested to be associated with uptake of Cl- but not Na+. Moreover, numerous multicellular complexes of MR cells developed in the chloride-free medium, suggesting to increase the apical surface area of MR cells for the enhancement of Cl- uptake. According to the results of double-labeling of Con A and Na+,K+-ATPase, the patterns of the relative abundance of MR cells labelled with Con A were similar to those examined by SEM, indicating that Con A is another suitable indicator to distinguish different types of MR cells. Western boltting analysis indicated that the amount of gill Na+,K+-ATPase was higher in the tilapia acclimated to F-Cl and H-Na than those acclimated to H-NaC1, suggesting that gill MR cells expressed various levels of Na+,K+-ATPase to perform the transport functions for different ions.