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研究生:林宓璇
研究生(外文):Mi-Hsuan Lin
論文名稱:研究褐藻醣膠中特定結構之生物活性
論文名稱(外文):Study on the Putative Structure and the Biological Functions of Fucoidan
指導教授:許先業楊文彬楊文彬引用關係
指導教授(外文):Hsien-Yeh HsuWen-Bin Yang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:英文
論文頁數:36
中文關鍵詞:褐藻醣膠轉化生長因子受器多醣體碳水化合物分析岩藻醣高效陰離子交換樹酯線性離子阱高效液相色譜法
外文關鍵詞:fucoidanTGF-β receptorspolysaccharidescarbohydrate analysisfucoseHPAECLTQ MSHPLC MS
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褐藻醣膠是一種水溶性水岩藻糖聚合而成且含有大量硫酸根鍵結的複雜多醣體。先前實驗室的研究中,我們已證實褐藻醣膠具有抑制肺癌及乳癌細胞生長的功效。在此,我們主要探討褐藻醣膠的化學成分與抗癌活性之相關性。將褐藻醣膠經由0.22 μm濾膜過濾後,經高效液相色譜法(HPLC)分析,發現過濾後的褐藻醣膠平均分子質量估計約500 Da,並定義其為小分子褐藻醣膠(LMF)。進一步探討LMF的岩藻糖含量,經由高效陰離子交換樹酯(HPAEC)分析,我們發現岩藻糖從LMF的釋放含量會隨著溫度水解處理時間增加而提升;以及經由不同濃度的醋酸再合併高溫水解處理後LMF釋放岩藻糖的含量也具有相同結果。我們進一步分析水解的LMF之抗癌功效,先前我們的研究證實,褐藻醣膠會促進癌細胞的轉化生長因子受器(TGFRs)降解並產生細胞自噬作用;在此,我們專注於LMF與水解後LMF 對於TGFRs的降解效率。研究結果顯示LMF可有效的促進TGFRs降解,然而水解後LMF無法有效率地促進TGFRs的降解,也無法抑制癌細胞生長。更進一步,我們利用質譜(ESI/MS)分析LMF內含成分,我們發現LMF內含硫酸根的岩藻糖,然而經過高溫與醋酸水解的LMF並無法測得富含硫酸根之岩藻糖。利用富含硫酸根的褐藻醣膠進一步分析細胞自噬作用,我們發現高度富含硫酸根之褐藻醣膠可以增強細胞自噬作用,而經水解後不含硫酸根之LMF無此功能。經由上述研究發現,我們推測褐藻醣膠的主要抗癌活性可能是來自於岩藻糖及其硫酸根離子的含量。
Fucoidan, a water-soluble and sulfated-fucans having complicated chemical structures, commonly found in brown seaweeds. Our previous studies demonstrated that fucoidan inhibits the growth of lung cancer cells and breast cancer cells. Here, we focus on the relationship between its chemical composition and in vitro anticancer activity. The major low molecular mass of fucoidan is named LMF and estimated average molecular mass about 500 Da by size-exclusion high-performance liquid chromatography (SEC-HPLC). To examine the content of fucose of LMF, we hydrolyzed the LMF via autoclave (121℃) and acetic acid. The residue was further applied the high-performance anion exchange chromatography (HPAEC) to identify the content of fucose from hydrolyzed LMF. We found that the released fucose from LMF was increased with a time-dependent manner by autoclave or a does-dependent of acetic acid in autoclave. Next, we examined the anti-cancer activity of the hydrolyzed LMF. Our previous studies demonstrated that fucoidan enhances transforming growth factor β (TGFβ) and its receptors (TGFRs) degradation in cancer cells. Here we found that LMF enhanced TGFRs degradation, whereas, the hydrolyzed LMF did not affect expression of TGFRs and decrease the viability of cancer cells. Furthermore, we examined the components of LMF by electrospray ionization mass spectrometry (ESI/MS), we found that the sulfated fucose group presented on the LMF. The results showed an essential factor in this study, the hydrolyzed LMF lost the sulfated group from fucose that the higher content of sulfated fucose group of LMF (SO3-LMF) induced the expression of LC3-II (an autophagy marker), but not hydrolyzed LMF. Taken together, we proposed that the bioactivity of fucoidan may be contributed from the content of sulfated fucose group on low molecular mass fucoidan.



Abstract 1
中文摘要 2
Introduction 3
Materials and Methods 5
Cell lines 5
Reagents and antibodies 5
Western blot analysis 5
Cell viability assay (MTT assay) 6
Partial acid hydrolysis 6
HPAEC analysis 6
Determination of cell morphology 7
Size exclusion of Fucoidan 7
LTQ analysis 7
Statistical analysis 8
Results 9
The size exclusion pattern for fucoidan of Hi-Q and of Sigma-Aldrich 9
Study on free form fucose released from low molecular mass of fucoidan (LMF) and the biological functions 9
The biological effects of LMF for anti-cancer in lung cancer cell 9
LMF reduces the expression of transformation growth factor receptor (TGFR) 10
LMF induces cell autophagy and apoptosis in lung cancer cells 10
Structure analysis of LMF 11
Discussion 13
References 15
Figure Legends 18
Figure 1. The size exclusion pattern for HQ-fucoidan and SA-fucoidan analyzed by HPLC 18
Figure 2. Free form fucose released from LMF and the biological functions 19
Figure 3. The effects of LMF on anti-cancers 23
Fiugre 4. LMF induced autophagy formation and apoptosis. 26
Figure 5. Structure analysis of SA-LMF by ESI/MS 30


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