3T3-L1 脂肪細胞培養及分化平台建立及其
應用於活性藻類的篩選

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洪綉茹(Hsiu-Ju Hung) 查詢紙本館藏

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論文名稱

3T3-L1 脂肪細胞培養及分化平台建立及其應用於活性藻類的篩選

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摘要(中)

本實驗建立3T3-L1前脂肪細胞培養及利用分化劑誘導其分化的機制，作為篩選藻類可能存在抑制脂肪細胞分化活性物質的實驗工作平台。利用黃豆異黃酮genistein 100 ?M ( = 27 ?g/ ml)為正反應對照組，分別測試藍綠藻的NP01、NP02、NP03、CR02、FE01、FE02、FE03，紅藻的Ｈa01、Gc01、Grs04、Pd01，及褐藻Ec01等12株不同藻細胞的甲醇萃出物在27、270和2700??g/ml的濃度下對3T3-L1前脂肪細胞株的分化抑制活性。
將3T3-L1前脂肪細胞培養於含DMEM-FBS的24孔培養盤，待其長滿後兩天，在培養液中添加insulin, dexamethasone和3-isobutyl-1-methylxathine作為分化誘發劑，隨後每兩日更換含有insulin的新鮮培養液至接種後的第十四天，分析其脂肪含量，而分化抑制劑與藻類萃出物的添加處理設定於接種後第六天。利用Oil redＯ染色法，比較各實驗組經分化劑誘導後之細胞培養，其細胞中脂質含量的差異，結果並未能發現測試的各藻株甲醇萃出物含有類似genistein之分化抑制效果，而且也不具備強化或減低genistein之抑制細胞脂質含量的作用。同時也發現實驗所採的各藻株甲醇萃出物，除FE02有稍許抑制細胞生長的活性外，並不會影響細胞的存活率。雖
然本研究未能發現到具有抑制脂肪細胞分化的藻株萃出物，但已能充分掌握此細胞分化培養的實驗條件，並證明這樣的實驗平台可應用於天然物對脂肪細胞分化抑制活性篩選與測試。

摘要(英)

The study tried to establish a 3T3-L1 preadipocyte culture and differentiation system for the screening of inhibiting agents of lipid accumulation in algal natural products. Inoculated preadipocytes were growing in 24-well plate of FBS-DMEM medium. After reaching confluent, culture medium were added insulin, dexamethasone and 3 isobutyl-1-mehtyl-xathine, and refreshed with new the medium containing insulin every two days afterwards. This culture system used 100 ?M genistein (27??g/ml) as positive control in comparison with the MeOH extracts of the algal cells including NP01, NP02, NP03, CR02, FE01, FE02, and FE03 of blue green algae, Ha01, Gc01, Grs04 and Pd01 of red algae, and Ec01 of brown algae. Concentrations of the algal extract applied for the screening were 27, 270 and 2700??g/ml. Lipid contents in the differentiated cells were analyzed by Oil red O staining. All the algal extracts tested were found to be of no effect like genistein in inhibition of cell differentiation and lipid accumulation. It was also found that the algal extracts showed no cytotoxicity or growth inhibition to the cells, except FE02 that showed slight growth inhibition to the cells but no cytotoxicity. Although we did not find any inhibition activity of lipid accumulation in the algal extracts, we found this culture and differentiable system of 3T3-L1 cell line were reliable and practicable for the screening of anti-obesity natural products.

論文目次

中文摘要 I
英文摘要 II
縮寫對照表 III
第一章前言
1.1 脂肪細胞的培養與分化 1
1.1.1 3T3-L 前脂肪細胞的培養 1
圖1.1 分化劑中三種成份: DEX, IBMX 和INS 之結構圖 2
1.1.2 脂肪細胞分化之轉錄因子 5
1.1.3 脂肪細胞分化的抑制 10
1.2 研究動機與目的 13
表1.1 世界衛生組織根據肥胖程度所制定的BMI 指標 14
第二章材料方法
2.1 儀器與藥品 17
圖 2.1 Genistein, oil red O, CBG-250 和MTT 之結構圖 19
2.2 3T3-L1 脂肪細胞株培養與分析 20
2.3 藻類的培養與萃取 24
圖 2.2 藻類100 ml, 1L 和20L 的培養模式 25
表 2.1 藻類的種名和來源 27
表 2.2 IBI 藻類培養液配方 28
第三章結果與討論 29
3.1. 3T3-L1 脂肪細胞生長分化條件的建立 29
3.1.2. d-Biotin 的補充添加對細胞分化之影響 32
3.1.3. Insulin 對細胞分化影響之測試 33
3.2. 抑制3T3-L1 細胞分化之正對照組genistein 之測試 34
3.3. 藻類萃出物對前脂肪細胞毒性的分析 34
3.4. 藻類萃出物對細胞內脂質含量的分析 35
圖3.1. 3T3-L1在day6時添加和不添加分化劑下培養至day15
的生長曲線 37
圖3.2. 不同培養時期的脂肪細胞形態和細胞內脂質生成的
顯微觀察 38
圖3.3. 不同培養時期的脂肪細胞經oil red O染色後之比較 38
圖3.4-A 細胞在不同濃度d-Biotin下經oil red O 染色後之比較 39
圖3.4-B 添加不同濃度d-Biotin培養至day14細胞脂質含量之比較39
圖3.5-A 細胞在不同濃度Insulin下培養至day14經oil red O染色 40
後之比較
圖3.5-B 添加不同濃度Insulin培養至day14細胞脂質含量的比較 40
圖3.6-A 細胞在不同濃度genistein 下其oil red O 染色之觀察 41
圖3.6-B 不同濃度之genistein 對脂肪細胞脂質堆積的影響 41
圖3.7 不同濃度DMSO 下細胞存活率之測試 42
圖3.8-A~L 藻類萃出物對脂肪細胞之毒殺性試驗 43
圖3.9-A~L 藻類萃出物對細胞內脂肪含量的影響 48
第四章討論 53
第五章參考文獻 55
附錄 61

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指導教授

周宏農(Hong-Nong Chou)

審核日期

2005-12-8

推文