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研究生:黃朝祿
研究生(外文):Chao-Lu Huang
論文名稱:廣鹽性吳郭魚適應不同高張環境時鰓上Na+,K+-ATPase表現與MR細胞型態之變化
論文名稱(外文):Changes in expression of Na+, K+-ATPase and morphometry of mitochondrion-rich (MR) cells in gills of euryhaline tilapia acclimated to various hypertonic environments
指導教授:李宗翰李宗翰引用關係
指導教授(外文):Tsung-Han Lee
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:81
中文關鍵詞:吳郭魚NKA滲透壓MR細胞
外文關鍵詞:Oreochromis mossambicusNa+K+-ATPaseOsmolalityMR cellGill
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本研究目的為瞭解廣鹽性吳郭魚(Oreochromis mossambicus)在面臨不同高滲透壓環境刺激時,鰓上Na+, K+-ATPase(NKA)的表現情形和生理變化的關係,本實驗利用不同高張環境馴化過程:(1)淡水轉移至半淡鹹水、(2)半淡鹹水轉移至海水、(3)淡水轉移至海水,使用形態、生理及生化、分子生物學的方法觀察及分析鰓和血清樣本。實驗結果顯示:(1)淡水轉移至半淡鹹水的氯離子、鈉離子濃度及滲透壓均在轉移後3至12小時間有明顯的增高;而鰓上NKA的活性和蛋白質表現量在轉移後12小時顯著上升;NKA的mRNA含量在轉移後6小時顯著上升;而以掃描式電子顯微鏡觀察鰓表皮,發現負責離子調控的MR細胞開口數量在3小時後會顯著減少在48小時後才會升高至未轉移前相當數量。(2)半淡鹹水轉移至海水的氯離子濃度無顯著差異,而滲透壓和鈉離子在1及6小時後有明顯的增高;NKA活性會在6小時後顯著降低,而在18小時後才會升高至未轉移前的值;而NKA蛋白質含量在長期適應後才有明顯增加;NKA mRNA含量卻無明顯變化;而MR細胞開口數量在3小時後會顯著減少在18小時後才會升高至未轉移前相當數量。(3)淡水轉移至海水的滲透壓、氯離子和鈉離子在1、1及4小時後有明顯的增高;NKA mRNA和蛋白質含量無明顯變化,活性在3小時後顯著降低;而MR細胞開口數量在1小時後即顯著減少。由上述實驗結果可知,當吳郭魚長期適應在不同鹽度環境時,一旦遭遇更高張鹽度刺激時,由MR細胞型態和開口數目、血液分析和NKA表現的差異性,顯示為適應不同的高張環境,所表現出的短期調節機制是完全不同的。
The purpose of the present study is to examine the expression of branchial Na+, K+-ATPase (NKA) in tilapia (Oreochromis mossambicus) upon hyperosmotic challenge. The present study including some biochemical and ultrasturctural examinations were done to compare the differences between fish transfered (1) from fresh water (FW) to brackish water (BW); (2) from BW to seawater (SW); and (3) from FW to SW. The results revealed that: (1) in blood, the osmolality and Cl- concentration significantly increased within 3 hrs after transfer from FW to BW. Significant increase of the Na+ concentration occurred within 12 hrs. In gill, NKA a1-isoform mRNA amount increased significantly within 6 hrs. Protein abundance and activity significantly elevated within 12 hrs. density decreased significantly within 3 hrs after transfer, then it increased again within 2 days after transfer; (2) in blood, the osmolality significantly increased within 1 hr after transfer from BW to SW. The Na+ concentration significantly elevated within 6 hrs. No significant difference was found in the Cl- concentration after transfer. In gill, NKA a1-isoform mRNA amount showed insignificant difference after transfer. Protein abundance increased significantly in long-term acclimation. NKA activity decreased significantly within 6 hrs, then it increased again within 18 hrs after transfer. MR cell density decreased significantly within 3 hrs after transfer, then it increased again within 18 hrs after transfer; (3) in blood, the osmolality and Cl- concentration significantly increased within 1 hr after transfer from FW to SW. The Na+ concentration significantly increased within 4 hrs. In gill, NKA a1-isoform mRNA amount and protein abundance showed insignificant difference after transfer. NKA activity decreased significantly within 3 hrs. Density of MR cells decreased dramatically in 1 hr. Taken together, changes in expression of NKA and morphometry of MR cells densities revealed that different mechanisms were exhibited in tilapia acclimated to varied hypertonic environments.
中文摘要 …………………………………………………………… 1
英文摘要 …………………………………………………………… 3
前言 ………………………………………………………………… 5
一、廣鹽性魚類 …………………………………………………… 5
二、Na+, K+-ATPase(NKA)……………………………………… 5
三、硬骨魚類的滲透壓調節機制 ………………………………… 7
四、廣鹽性魚類適應海水過程 …………………………………… 9
五、廣鹽性魚類鰓表皮細胞分布、結構與離子調節 …………… 11
六、研究目的 ……………………………………………………… 11
材料與方法 ………………………………………………………… 14
一、實驗動物 ……………………………………………………… 14
二、馴化環境鹽度的配製 ………………………………………… 14
三、實驗設計 ……………………………………………………… 14
(一)預備實驗 …………………………………………………… 14
(二)由淡水轉移至半淡鹹水馴化 ……………………………… 15
(三)由半淡鹹水轉移至海水馴化 ……………………………… 15
(四)由淡水轉移至海水 ………………………………………… 15
四、實驗方法 ……………………………………………………… 16
(一)採血 ………………………………………………………… 16
1. 氯離子濃度的測定 ………………………………………… 16
2. 鈉離子濃度的測定 ………………………………………… 17
3. 滲透壓測定 ………………………………………………… 17
(二)取鰓 ………………………………………………………… 17
1.NKA活性的測定 …………………………………………… 17
2.NKA蛋白質定量 …………………………………………… 21
3.NKA mRNA定量 …………………………………………… 21
4.掃描式電子顯微鏡觀察……………………………………… 25
五、統計分析方法 ………………………………………………… 26
結果 ………………………………………………………………… 27
一、由淡水轉移至半淡鹹水馴化(由0‰ 至20‰):生存 …… 27
(一)血液的分析 ………………………………………………… 27
(二)鰓上NKA的表現…………………………………………… 27
(三)鰓絲上MR細胞的開口數量及型態 ……………………… 28
二、由半淡鹹水轉移至海水馴化(由20‰ 至35‰):生存…… 28
(一)血液的分析 ………………………………………………… 28
(二)鰓上NKA的表現…………………………………………… 28
(三)鰓絲上MR細胞的開口數量及型態 ……………………… 29
三、由淡水轉移至海水(由0‰ 至35‰):死亡 ……………… 29
(一)血液的分析 ………………………………………………… 29
(二)鰓上NKA的表現…………………………………………… 30
(三)鰓絲上MR細胞的開口數量及型態 ……………………… 30
討論 ………………………………………………………………… 31
參考文獻 …………………………………………………………… 38
表與圖 ……………………………………………………………… 50
附錄 ………………………………………………………………… 75
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