重金屬鋅或鎘誘發海葵(Aiptasia pulchella)體內金屬硫蛋白之研究

• Main Authors: 黃嘉儀, Huang, Chia-I
• Other Authors: 陳俊宏, 臺灣大學：動物學研究研究所
• Format: Thesis
• Language: English
• Published: 2007
• Subjects: 金屬硫蛋白; 海葵; 鋅; 鎘; 誘發性; 免疫組織染色; metallothionein; sea anemone; Aiptasia pulchella; zinc; cadmium; Polar Biology
• Online Access: http://ntur.lib.ntu.edu.tw/handle/246246/56679; http://ntur.lib.ntu.edu.tw/bitstream/246246/56679/1/ntu-96-R94b41030-1.pdf

金屬硫蛋白 (Metallothionein, MT)是一種含有大量cysteine、耐熱的小分子量蛋白質。這種蛋白質已經被證實可以和重金屬連結，除了可以調控多餘的必需重金屬鋅或銅外，若是有非必需的重金屬（鎘或汞）進入生物體內，金屬硫蛋白可與這些金屬離子結合，作為解毒機制的一環。從之前的文獻知道，metallothionein具有作為生物指標的潛力。本實驗的實驗動物，海葵Aiptasia pulchella 容易飼養並具有無性生殖的特性，可減少實驗中的個體差異。將實驗動物暴露在不同濃度的鋅溶液 (0, 0.25, 0.5 或1.0 mg/L) 24, 48 或96小時 以及不同濃度的鎘溶液（0, 0.05 ,0.25 或 0.5 mg/L) 24, 48 或72 小時，海葵的外型會有顯著的變化：身體和觸手會縮小，並且會有變黑的情形發生。在檢測海葵組織內重金屬的濃度，可以發現組織內重金屬的濃度會隨著處理時間或是重金屬濃度的增加而增加。透過抗體辨識發現海葵的metallothionein的分子量應該位在11~17 kDa之間，主要表現的至少有三種不同大小的蛋白質，推測其中之一應該為constitutive metallothionein；另外的為誘發性的metallothionein。誘發性的metallothionein的量會隨著暴露的時間以及金屬濃度增加，其表現量會有增加的趨勢。根據免疫組織染色的結果，Metallothionein在海葵體內呈現不均勻分佈。當暴露在較高濃度的鋅或鎘溶液中，身體部分的亮點會明顯增加；顯示metallothionein在身體部位呈點狀且較均勻地分佈。 Metallothioneins (MTs) are cysteine rich, heat stable and low molecular weight proteins. They have been widely used as biomarkers to reflect the level of heavy metal pollution. Sea anemones (Aiptasia pulchella) should reflect the real condition of its surroundings due to its sedentary. Normally, they are asexually reproduced by pedal laceration in laboratory cultural system. After exposure to different concentrations of cadmium (0, 0.05, 0.25 or 0.5 mg/L for 24, 48 or 72 hours) or zinc (0, 0.25, 0.5 or 1.0 mg/L for 24, 48 or 96 hours), obvious morphological alterations in A. pulchella were recorded. Their tentacles turned dark and their body shrank. By using atomic absorption to measure Zn or Cd concentration in A. pulchella, those metal-exposed sea anemones showed a time-dependent and a dose-dependent both zinc and cadmium accumulation. From the results of immunoblotting, the amount of metallothioneins in those metal-exposed A. pulchella increased following by a time-and dose-course. The molecular weight of the detected MTs was between 11 and 17 kDa. There were at least three bands appeared. One is assumed to be constitutive MT and the others are assumed to be induced metallothioneins. Immunofluorecent antibody detection of the distribution of metallothionein in A. pulchella under zinc or cadmium treatment demonstrated that the fluorescent particles in the body column significantly increased and the fluorescence of tentacles also intensified. The induction of metallothioneins in A. pulchella was shown being responsive to different concentrations of metals. Contents 口試委員審定書 --- i Abstract (English) --- ii Abtract (Chinese) --- iii 1. Introduction 1.1. Characteristics of metallothioneins --- 1 1.2. Heavy metals --- 3 1.3. Heavy metal pollution in the coastal areas --- 4 1.4. Cnidarians and heavy metals --- 5 1.5. Metallothionein isoforms --- 6 1.6 Toxicity assessment --- 7 2. Materials and Methods 2.1. Animals --- 10 2.2. Zn or Cd exposure --- 10 2.3. Measurement of heavy metal bioaccumulation --- 11 2.4. Partial purification of Metallothionein --- 11 2.5. SDS-PAGE --- 12 2.6. Immunoblotting --- 12 2.7. Immunohistochemistry --- 13 2.8. Statistics --- 14 3. Results 3.1. Morphological change after exposure to zinc and cadmium --- 15 3.2. Bioaccumulation in A. pulchella --- 15 3.3. Electrophoretic studies --- 16 3.4. Immunoblotting --- 17 3.5. Immunohistochemistry (IHC) --- 17 4. Discussion --- 18 5. References --- 24 FIGURES AND TABLES Fig 1. Morphological change after Zinc exposure --- 32 Fig 2. Morphological change after Cadmium exposure --- 34 Fig 3. Bioaccumulation of Zinc in Aiptasia pulchella --- 36 Fig 4. Bioaccumulation of Cadmium in Aiptasia pulchella --- 37 Fig 5. One-dimensional SDS-PAGE after Zinc exposure --- 38 Fig 6. One-dimensional SDS-PAGE after Cadmium exposure --- 40 Fig 7. Immunoblotting of Metallothionein in A. pulchella after Zinc exposure --- 42 Fig 8. Immunoblotting of Metallothionein in A. pulchella after Cadmium exposure --- 43 Fig 9. Metallothionein distribution in A. pulchella after Zinc exposure --- 44 Fig 10. Metallothionein distribution in A. pulchella after Cadmium exposure --- 49 Table 1. Zinc concentration in tissues of A. pulchella --- 54 Table 2. Cadmium concentration in tissues of A. pulchella --- 55