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研究生:梁愛羚
研究生(外文):Ai-Ling Liang
論文名稱:探討藻褐素在不同溶劑系統中的穩定性以及在兩種肝細胞株中的吸收
論文名稱(外文):The stability of fucoxanthin in different solvent systems and the cellular uptake in SK-Hep-1 and BNL CL.2 cells
指導教授:胡淼琳胡淼琳引用關係
指導教授(外文):Miao-Lin Hu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:85
中文關鍵詞:藻褐素穩定性細胞吸收肝細胞
外文關鍵詞:fucoxanthinstabilityuptakeliver cells
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  本實驗之目的乃是在探討藻褐素在培養基中的穩定性,及兩種肝細胞(BNL CL.2正常肝細胞和SK-Hep-1肝癌細胞)的吸收率。藻褐素先以絕對酒精配製成10 mM,再以酒精將藻褐素稀釋成1.0 mM(stock A)或以100%胎牛血清稀釋成1.0 mM(stock B)。取10μL Stock A加到10 mL含有10%FBS的DMEM培養基(A1),或不含FBS的DMEM中(A2)。另取10μL Stock B加到10 mL含有10%FBS的DMEM培養基(B1),或不含FBS的DMEM中(B2)。各組藻褐素的終濃度均為10μM。
  結果顯示:培養3、6、12、24、48、72小時之後,藻褐素會因培養的時間增加而逐漸降解;其中以A2組的穩定度最好,在經過了72小時後仍有73.3%的藻褐素未被降解。而B1組的降解速度最快,於72小時培養後只有約34.9%的藻褐素未被降解。
  在SK-Hep-1肝癌細胞吸收率方面,培養24小時後,藻褐素吸收率達到最高,其中以A2組的細胞9.81%吸收量最高,而以A1組的細胞2.10%吸收率最低。以BNL CL.2 而言,於培養24小時之後,以B1組的5.60%吸收率最高,以A1組的2.91%吸收率最低。在細胞培養過程中,HPLC分析圖出現一個不明的藻褐素代謝產物;此物質也會隨著時間的增加而累積量增加。在SK-Hep-1細胞培養中,此未知物於培養24小時之後,其累積量達最高。對BNL CL.2細胞而言,此一未知物質則是在48小時達到最高量。  
  此外,本研究也觀察藻褐素對細胞型態的影響;結果顯示:隨著培養時間的增加,SK-Hep-1肝癌細胞型態逐漸變形和細胞膜變得不完整,尤其在不含血清的培養基培養下,細胞的存活率很低;而BNL CL.2細胞無法在不含血清的培養基超過12小時長時間的培養,然而在含血清的培養基培養下,隨著培養時間的增加,細胞型態也會變形,並有囊泡產生的現象。
  以上的結果顯示:以酒精配製的藻褐素在不含血清的培養基中(A2組)穩定性較好。而且SK-Hep-1細胞在A2組對藻褐素的吸收也最佳。然而在BNL CL.2細胞中,則是以胎牛血清稀釋的藻褐素在含血清的培養基中(B1)的吸收率最好。
The purpose of this study was to investigate the stability of fucoxanthin in different solvent systems and the cellular uptake in two types of liver cells. A ethanol was used first to prepare a 10 mM fucoxanthin solution, which was then further diluted to 1.0 mM in ethanol (Stock A) or in 100% FBS (Stock B). A 10 μL Stock A was added to 10 mL DMEM medium containing 10% FBS (A1) or without containing FBS (A2). Likewise, A 10 μL Stock B was added to 10 mL DMEM medium containing 10% FBS (B1) or without containing FBS (B2). The final concentration of fucoxanthin in these solvent systems was 10 μM.
We found that the fucoxanthin in the four different solvent systems degraded with increasing incubation time (3-72h). Fucoxanthin in group A2 had the best stability, with 73.3% of fucoxanthin remaining at 72 h, while fucoxanthin in group B1 had the poorest stability, with only 34.9% fucoxanthin remaining at 72 h.
Regarding cellular uptake in SK-Hep-1 cells, fucoxanthin in all aolvent systems had the highest uptake at 24 h, at which time fucoxanthin in group A2 had the highest uptake (9.81%), while group A1 had the lowest uptake (2.10%). For BNL CL.2 cells, group B1 had the highest uptake (5.6%), while group A1 had the lowest uptake (2.91%) at 24 h. In the HPLC profile, there was an unknown peak, which was suspect to be a metabolic product of fucoxanthin by cells. The concentration of this product reached the maximum level at 24 h and 48 h of incubation for SK-Hep-1 cells and BNL CL.2 cells, respectively.
In addition, we noted fucoxanthin-induced changes in cell morphology. With the increase in incubation time, SK-Hep-1 cells became deformed and had low viability, especially when incubated in the FBS-free medium (groups A2 and B2). Fucoxanthin also affected the morphology of BNL CL.2 cells by causing deformation and production of vesicles, even when FBS was present (groups A1 and B1).
In summary, this study shows that the stability was better when the fucoxanthin stock was prepared in ethanol and then added into the FBS-free medium (group A2), which also has the best cellular uptake of fucoxanthin in SK-Hep-1 cells. In BNL CL.2 cells, however, cellular uptake of fucoxanthin is better in B1 group, which was prepared in 100% FBS and then diluted in DMEM medium containing 100% FBS.
目次 I
表目錄 IV
圖目次 V
縮寫表 VII
中文摘要 VIII
英文摘要 X
壹、前言 1
貳、文獻整理 5
一、類胡蘿蔔素(CAROTENOIDS) 5
二、藻褐素(FUCOXANTHIN) 7
1、藻褐素的結構 8
2、藻褐素的代謝 8
3、藻褐素生理活性 9
三、載體運送 10
1、微粒體-Micelles 11
2、脂質小體-Liposomes 12
3、甲基-β-環狀糊-Methyl-beta-cyclodextrin(M-β-CD) 12
叁、研究動機與目的 14
一、研究動機 14
二、研究目的 15
肆、實驗架構 16
伍、材料與方法 17
一、實驗試劑 17
二、細胞選定 17
三、細胞解凍 18
四、細胞培養 18
五、細胞保存 18
六、藻褐素之載體製備 18
七、細胞存活率分析-細胞計數 19
八、穩定性分析 19
九、細胞吸收 19
十、萃取與HPLC分析 20
1、培養基的穩定性萃取 20
2、萃取細胞內的藻褐素 20
3、HPLC 分析 21
十一、統計分析 22
陸、結果與討論 24
一、藻褐素在不同系統製備中的穩定性 24
二、細胞生長曲線 36
三、SK-HEP-1 CELLS對於藻褐素的吸收 51
四、BNL CL.2. CELLS對於藻褐素的吸收 68
柒、結論 78
捌、文獻參考 79
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