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研究生:陳怡靜
研究生(外文):I-Ching Chen
論文名稱:花笠螺齒舌礦化─鐵運輸機制之探討
論文名稱(外文):Biomineralization in the Radula of the Limpet Cellana toreuma--the Mechanism of Iron Transportation
指導教授:李家維李家維引用關係
指導教授(外文):Chia-Wei Li
學位類別:碩士
校院名稱:國立清華大學
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:2002
畢業學年度:90
語文別:中文
論文頁數:75
中文關鍵詞:花笠螺齒舌電子顯微鏡軟體動物
外文關鍵詞:limpetradulaironTEM
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軟體動物的齒舌是研究鐵與矽的生物礦化過程的好材料,笠螺的齒舌含有結晶態的鐵(goethite)及非結晶態的矽(opal),由於刮食的過程中會造成牙齒的磨損,笠螺會不斷地造新牙,齒舌的生長點先在新牙腔內佈建規則排列的有機質絲,並在不同的發育階段將鐵與矽精確地沈積在有機質架構上,以用來補充磨損的牙,使得整條齒舌的礦化成為一個線性的動態平衡。
本研究以台灣東北角石門一帶的花笠螺 cellana toreuma為材料,對其齒舌礦化階段中的stageⅠ、stageⅡ以及生長點進行組織切片、SEM及TEM觀察,除了印證齒舌在每個礦化階段所發生的變化,及鐵的運輸途徑之外,我們發現了一個新的含鐵組織,這個組織從生長點延伸出來覆蓋在整條齒舌的上皮細胞外,組織內的細胞分佈稀疏,細胞內有由ferritin組成的鐵囊胞。在stageⅡ的此組織內,細胞間質則充滿了直徑30-50 nm的鐵結晶體,此為先前的研究不曾發現過的。
而在生長點中已經可以看到牙齒的雛形,牙腔與牙基有明顯的界線區隔。牙齒旁的細胞可分為兩大類,牙腔旁的細胞具有微絨毛,負責將物質運送到牙腔中,但與其他stage中牙冠旁具有微絨毛的上皮細胞不同的是這些細胞具有tight junction;而牙基旁的另一群細胞內則佈滿了桑椹般的深色球囊,這也是先前的研究不曾發現過的,我們推測這些球囊與牙齒的形成必定有著重要的關連。
另外,我們更進一步分析齒舌內的蛋白質,找到一個在牙齒、上皮細胞與周邊組織都存在的蛋白質,SDS-PAGE上顯示分子量約為16 kDa,質譜儀分析其實際分子量為11.558 kDa。由胺基酸組成分析,我們估算其pI值約5.01。以兔子誘發多株抗體從事TEM切片上的免疫染色,結果在牙冠/牙基/上皮細胞/周邊組織的免疫膠體金顆粒密度比約為16/10/4/5,顯示這個蛋白質主要表現在牙腔與牙基內。藉由這些分析,我們推測這個蛋白質為參與花笠螺齒舌礦化的酸性醣蛋白。

The radula of Mollusca is an excellent material to investigate the biomineralization process of iron and silica in living organism, because there is crystalline goethite and amorphous opal in the limpet’s radula. Because of the wearing of teeth by scraping on the rock, the limpet secretes organic matrix from the sac and deposits iron and silica precisely on the organic matrix framework through different stages. Thus, the process of radula biomineralization is in a linear and dynamic balance.
The limpet Cellana toreuma was collected from the coast of Shi-man. We used tissue section, scanning electron microscope (SEM) and transmission electron microscope (TEM) to investigate the biomineralization in stage I, stage II and radular sac of the limpet. A new tissue containing iron was found. This tissue extends from the sac and covers the superior epithelial cells of the radula. Cells with sidersomes constructed by ferritin particles distribute sparsely in the tissue. In stage II, the cellular matrix of the tissue is filled with iron crystals that are about 30-50 nm in diameters. The existence of this tissue is an observation which has not been reported previously.
In the radular sac, we observed newly formed teeth, and a clear boundary between the cusp and the base. There are two types of cells near the newly formed teeth. The cells adjacent to the cusp have microvilli and may function in transferring materials into the cusp. Some of these cells have tight junctions and differ from the cells with microvilli in stage II. The other type of cells adjacent to the base contains mulberry-like cysts which show high electron density. These newly observed cysts are proposed to be related to the forming of the tooth.
Furthermore, we analyzed the proteins in the radula, and found one protein expressed not only in the tooth, but also in the superior epithelial cells and in the peripheral tissue. By amino acid composition analysis, its pI value is about 5.01. There was a difference of molecular weight using different analytical approaches. Its molecular weight in SDS-PAGE is about 16 kDa, in mass spectrum is 11.558 kDa and we propose the difference may due to glycosylation. Antibodies were generated from a rabbit. Using TEM and immunogold staining techniques we found that the ratio of immunogold density in cusp /base /superior epithelial cell /peripheral tissue is about 16/ 10/ 4/ 5, showing that the protein is predominantly expressed in the cusp and the base. Thus, we propose it should be an acid glycoprotein associated with the biomineralization process in the limpet’s radula.

Ⅰ 中文摘要 Ⅰ
Ⅱ 英文摘要
Ⅲ 誌謝 Ⅱ

Ⅳ 目錄 Ⅳ
一﹑前言 1
二﹑材料與方法 7
1. 採樣
2. 組織切片
3. 光學顯微鏡觀察 7
7
7
4. 掃描式電子顯微鏡 (SEM) 樣本製備
(1) 齒舌外型觀察 8
8
(2) 能量分散光譜分析 8
5. 穿透式電子顯微鏡 (TEM) 樣本製備 8
(1) 齒舌切片形態觀察 8
(2) 能量分散光譜分析 9
6. 耦合電漿質譜分析 (ICP-mass) 樣本製備 9
7. 花笠螺齒舌蛋白質萃取 10
8. 蛋白質膠體電泳分析 10
9. 16 kDa蛋白質抗原製備 11
10. 16 kDa蛋白質抗體製備 11
11. 西方墨點分析 (Western blotting) 11
12. 免疫膠體金染色 (Immunolgold staining) 12
13. HPLC 分離16 kDa蛋白質 13
14. 質譜儀分析前處理 13
15. 16 kDa胺基酸組成分析 13
16. 電子順磁共振光譜分析 13
三﹑結果 15
1. 花笠螺的生長環境 15
2. 齒舌型態觀察 15
3. 齒舌中鐵的分佈 17
4. 齒舌中矽的分佈 19
5. 齒舌生長點的切片觀察 19
6. 蛋白質篩選,抗體測試與免疫膠體金染色 20
(1) 齒舌中蛋白質的分佈 20
(2) 抗體測試 20
(3) 免疫膠體金染色 20
(4) HPLC分離16 kDa蛋白質與質譜儀分析 21
(5) 16 kDa蛋白質的胺基酸組成與等電點(pI值)預測 21
四﹑討論
1. 齒舌礦化 22
(1) 耦合電漿質譜分析 (ICP-mass) 22
(2) 齒舌生長點 22
(3) 牙基 23
(4) 牙冠與牙基交界處 (junction zone) 24
(5) 牙冠 25
2. 鐵的運輸 26
(1) 上皮細胞 26
(2) 周邊組織 26
3. 16 kDa蛋白質 28
五﹑結論 30
六﹑參考文獻 31
七﹑圖表 36
八﹑附錄 70

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