Galectin-1蛋白對細胞生長於幾丁聚醣膜上的影響及調控機制之研究

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姓名

張鈺媖(Yu-Ying Chang) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

Galectin-1蛋白對細胞生長於幾丁聚醣膜上的影響及調控機制之研究
(The study of mechanism of galectin-1 regulates cells proliferation on chitosan membrane)

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摘要(中)

幾丁聚醣是由螃蟹、龍蝦、蝦子這類甲殼類動物外殼中的幾丁質所衍生而來的。在臨床醫學上，幾丁聚醣膜已被使用當作具有半滲透性的生物敷料。雖然幾丁聚醣膜是不具有細胞毒性的，但有些細胞種類 (如: 3T3纖維母細胞)就是無法於幾丁聚醣膜表面進行貼附及增生的作用。近年有許多的研究已指出galectin-1 (Gal-1, 是一種?-半乳糖苷結合的蛋白)對於加速細胞貼附於細胞外基質及誘導細胞的增生而言，扮演重要的角色。因此，在本篇的研究中，我們將探討Gal-1蛋白影響3T3細胞在幾丁聚醣膜上生長的情形。發現當3T3細胞中的Gal-1蛋白被大量表現之下，無法促進細胞生長於幾丁聚醣膜上。然而，將Gal-1蛋白吸附在幾丁聚醣膜上時，則能明顯的加強細胞在幾丁聚醣膜上增生的效率。若處理Gal-1單株抗體及Thiodigalactoside (TDG, lectin的抑制劑)，則能抑制Gal-1對細胞增生的影響，此說明了確實是幾丁聚醣膜上所吸附的Gal-1蛋白對細胞的增生所產生的特殊影響。另外，利用免疫標定的方式也偵測到當3T3或CHOA細胞培養在Gal-1吸附的幾丁聚醣膜上時，會明顯的降低細胞內某些蛋白質酪胺酸磷酸化的表現；當使用NaF (絲胺酸/息寧胺酸去磷酸酶抑制劑)處理細胞，NaF明顯的抑制3T3細胞在Gal-1蛋白吸附的幾丁聚醣膜上的增生。此結果表示由Gal-1所誘導的細胞貼附及增生，主要是藉由改變蛋白質磷酸化所產生的訊號傳遞，以進而促進細胞在幾丁聚醣膜上的生長。

摘要(英)

Chitosan is a derivative of chitin extracted from the exoskeleton of lobsters, crabs and shrimps. In clinical medicine, chitosan membrane (a tissue-engineered chitin membrane) has been used as a semi-permeable biological dressing. Although the chitosan membranes showed no cytotoxicity, some cell types (e.g. 3T3 cell) were unable to attach and proliferate on the surface of chitosan membrane. A number of studies suggested that galectin-1 (Gal-1, a b-galactoside-binding protein) plays an important role in enhancing the adhesion of cells to extracellular matrix and inducing cell proliferation. Therefore, we here explored the applicability of Gal-1 on enhancing the growth of 3T3 cell on the surface of chitosan membrane. The results showed that over-expression of Gal-1 could not enhance 3T3 cell proliferation on chitosan membrane. However, coating the chitosan membrane with recombinant Gal-1 proteins significantly enhanced the efficiency of 3T3 cell proliferation. However, this effect was inhibited by Gal-1 monoclonal antibody and thiodigalactoside (TDG, a lectin inhibitor), suggesting the specific effect of Gal-1 on the proliferation of 3T3 cell. Moreover, immunoblotting detected a significant reduction in tyrosine phosphorylation of several proteins in 3T3 fibroblast and CHOA cell growing on Gal-1-coated chitosan membrane. The results also showed that NaF (a serine/threonine phosphatase inhibitor) dose-dependently inhibits 3T3 cell proliferation on Gal-1-coated chitosan membrane. These findings support a proposed role for altered levels of protein phosphorylation in Gal-1-mediated cell attachment and proliferation.

關鍵字(中)

★ 幾丁聚醣膜
★ Galectin-1

關鍵字(英)

★ Galectin-1
★ Chitosan membrane

論文目次

目錄 I
圖表目錄 V
縮寫表 VII
第一章緒論
第一節 Galectin-1的簡介 1
第二節幾丁質 (Chitin)及幾丁聚醣
(Chitosan)的簡介 2
第三節蛋白質磷酸化作用的角色 5
第四節文獻回顧 6
第五節研究動機與目的 6
5.1 研究動機 6
5.2 研究目的 7
第二章材料與方法
第一節實驗材料 8
1.1 實驗細胞株 8
1.2 實驗菌株 8
1.3 幾丁聚醣薄膜 (Chitosan membrane) 8
第二節 Gal-1轉殖細胞株的製備 9
2.1 質體DNA (pcDNA3.1-gal-1)的抽取與純化 9
2.2 質體pcDNA3.1-gal-1的轉殖 (Transfection) 10
2.2.1 細胞的培養 10
2.2.2 轉殖作用 10
2.3西方墨點法 (Western Blot) 11
2.3.1 細胞的收集 11
2.3.2 蛋白質的定量 11
2.3.3 細胞內Gal-1蛋白質表現量 12
2.4 免疫螢光法 (Immunofluorescence
Microscopy) 12
2.5 Gal-1轉殖細胞於幾丁聚醣膜上之生長 13
2.6 內生性Gal-1調控CHOA細胞於幾丁聚
醣膜上的增生 13
第三節 Gal-1蛋白質的純化 13
3.1 純化Gal-1 13
3.2 蛋白質的濃縮及冷凍乾燥 14
第四節 Gal-1吸附於幾丁聚醣膜 14
4.1 以交聯劑Carbodiimide接枝Gal-1
於幾丁聚醣膜上 14
4.2 自然吸附Gal-1幾丁聚醣膜上 15
4.3 檢測Gal-1於幾丁聚醣膜上吸附的程度 15
第五節 Gal-1對細胞生長於幾丁聚醣膜之影響 15
5.1 電子顯微鏡觀察細胞表面型態 (Morphology) 15
5.2 Gal-1單株抗體及TDG抑制3T3細胞於
幾丁聚醣膜上的增生 16
第六節　Gal-1在幾丁聚醣膜上對3T3細胞
增生所引起的訊號傳遞 16
第三章結果
1.內生性Gal-1蛋白質對細胞生長的影響 18
1.1 不同種細胞在幾丁聚醣膜上的生長情形 18
1.2 內生性Gal-1影響CHOA細胞於幾丁聚醣
膜上的增生 18
1.3 Gal-1穩定轉殖細胞株 (stable clone)的篩選19
1.4 Gal-1轉殖細胞於幾丁聚醣膜上的生長 19
1.5不同細胞外泌性Gal-1蛋白質的測定 19
2.外源性Gal-1蛋白質對細胞生長的影響 20
2.1 Gal-1 蛋白質於幾丁聚醣膜上吸附的程度 20
2.2 3T3細胞於Gal-1吸附之幾丁聚醣膜上的生長 20
2.3 電子顯微鏡觀察細胞表面型態 (Morphology) 21
2.4 Gal-1單株抗體及TDG抑制3T3於幾丁
聚醣膜上的增生 21
2.5 外源性Gal-1蛋白在幾丁聚醣膜上
對細胞增生所引發的訊號傳遞 22
第四章討論 24
第五章結論及展望 29
第六章參考文獻 43
附錄 50

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指導教授

黃榮南(Rong-Nan Huang)

審核日期

2003-7-11

推文