臺灣博碩士論文加值系統

蜂膠中的咖啡酸苯乙酯 (caffeic acid phenethyl ester，CAPE)，已被證實具有抗氧化、抗發炎、抗病毒、及抑制癌細胞生長等活性。本研究利用多株癌細胞測試 CAPE 的毒殺細胞作用，發現以終濃度 0-20 μg/mL 的 CAPE 處理癌細胞 HL-60、HepG2、C3A、CL1-0、H460、Caco2、MCF-7、PA-1，48 小時後，細胞之存活率降低，IC50 為 < 2 μg/mL 或介於 4 - 16 μg/mL 之間；但是，以鄉圖濃度的 CAPE 處理正常肺細胞 WI-38 及血球細胞 PBMC，48 小時後，細胞的存活率與控制組相似，IC50皆 > 20 μg/mL。以 15 μg/mL CAPE 處理 CL1-0 細胞 48 小時後，發現於細胞的細胞質有類似細胞自噬體 (Autophagosome) 的囊泡 (vacuole) 出現。利用西方雜配來偵測細胞自噬特徵 LC3-II 蛋白的表現，發現以 15 μg/mL 的 CAPE 處理 CL1-0，隨著處理時間由 0 小時增長到 48 小時，LC3-II 蛋白的表現會上升約 5 倍，表示確有細胞自噬的發生。

本研究亦證明由三合春生技公司的豆類發酵液-優豆靈液 (U-lifespring , ULS) 上清液，能夠抑制老鼠巨噬細胞 Raw 264.7 因 LPS 處理而誘發的一氧化氮 (nitric oxide, NO)的生成，並且此抑制作用為 dose-dependent，也發現 ULS 上清液，能夠使人類肝癌細胞 HepG2死亡。將 ULS 上清液，以等體積的乙酸乙酯 (ethyl acetate，EA)做二次液相-液相劃分，發現二次EA 層具有抗發炎活性及毒殺細胞株 CL1-0、A549、WI-38 的活性，水層則不具有此二活性。進一步利用正向 HPLC 對二次EA 層做分離及純化，發現所分離出來的 13 個分離 fraction ，其中 fraction 10 能夠抑制 Raw 264.7 細胞因 LPS 刺激而產生的一氧化氮達 59%。將 fraction 10 以質譜儀進行身分鑑定，發現 fraction 10 具有一分子量為 345.33 g/mole 的化合物。

Caffeic Acid Phenethyl Ester (CAPE) is found in resinous mixture used by honey bees, commonly called propolis and the CAPE has been shown to have antioxidant, anti-inflammatory, anti-viral and is cytotoxic to cancer cells. The effect of CAPE to kill many strains of cancer cells is studied. It has been found out through experiments that the concentration of 0-20 μg/mL of CAPE to the HL-60, HepG2, C3A, CL1-0, H460, Caco2, MCF-7, PA-1 cancer cell lines showed decreased cell survival after 48 hrs. The Inhibition concentration (IC50) was <2 µg/ml or 4 – 16 µg/mL according to the cell lines. The concentration of CAPE was also used in normal lung WI- 38 cells and normal blood PBMC cells, in 48 hrs, the cell survival and the control group showed >20 µg/mL. CL1-0 cell line was treated with 15 µg/mL of CAPE for 48 hrs. The morphology of the cell was examined using Fluorescence Inverted Microscope (Axiovert 200) and showed vesicles in the cell which may cause in autophagy. Western blotting was done to check the expression of LC3-II protein during autophagy and found out that 15 µg/mL of CAPE from 0 hrs to 48 hrs showed the gradual increase in expression of LC3-II protein and hence the autophagy occurs.
This study showed the use fermented legume mixture (U-lifespring, ULS) to examine the anti-inflammatory response against Raw 264.7 macrophage. The ULS supernatant has shown to inhibit the Raw 264.7 mouse macrophages, which contains nitric oxide induced by the LPS treatment. This inhibition is dose dependent manner. The ULS supernatant also showed inhibition against human hepatoma HepG2 cells. Equal volumes of ethyl acetate (EA) were added to the ULS supernatant which is called EA partition. The layer containing the EA has anti-inflammatory activity and showed cytotoxic effect against CL1-0, A549, WI-38 cell lines whereas the water layers do not have any activity. Secondary separation and purification of the EA layer was done using HPLC and isolated 13 separate fractions. The fraction 10 inhibited nitric oxide in the Raw 264.7 cells stimulated by LPS up to 59%. Mass spectrometry was used to identify the fraction 10 and found out that the molecular weight is 353.33 g/mole of the compound.

中文摘要 i
Abstract ii
表目錄 vii
圖目錄 viii
前言 1
目的 7
材料 8
I. 藥品配製及溶劑 8
II. 豆類發酵液 8
III. 細胞株及培養細胞用之培養基 8
方法 10
I. 細胞培養 10
A. Raw264.7 細胞之繼代培養、凍存及解凍方法 10
B. 貼壁型細胞之繼代培養、凍存及解凍方法 11
C. 懸浮型細胞之繼代培養、凍存及解凍方法 11
D. 混合型細胞之繼代培養、凍存及解凍方法 12
E. 人類周邊血液 (peripheral blood monouclear cells ,PBMC) 之分離 15
F. 細胞計數 15
II. 以 MTT assay 試驗 CAPE 對細胞之毒殺作用 15
A. 貼壁型細胞 15
B. 混合型細胞 16
C. 懸浮型細胞 16
D. 數據分析 16
III. 蛋白操作技術 17
A. 蛋白定量 17
B. 15% SDS-PAGE 17
C. 西方雜配 (Western blot) 17
D. TCA沉澱 18
E. Tricine SDS-PAGE 18
F. 銀染 19
G. 蛋白質鑑定 19
IV. 細胞自噬之偵測 19
V. ULS 上清液萃取與純化及化學成分鑑定 20
A. 豆類發酵液上清液製備 20
B. ULS/ddH2O 溶液製備 20
C. ULS 上清液分離 20
D. 化學物鑑定 22
VI. 抗發炎活性試驗 23
A. 一氧化氮生成率測定 23
B. 待測樣品對Raw 264.7的毒性試驗 23
結果 25
一、CAPE 25
I. CAPE 對 10 株癌細胞株及 2 株正常細胞株之毒殺能力 25
II. CAPE 造成 CL1-0 細胞細胞自噬 25
二、ULS 26
I. ULS上清液的抗發炎及抗癌活性 26
II. 分析 ULS 中抗癌與抗發炎活性成分分析 26
A. 以 TCA 濃縮分析蛋白 26
B. 以冷凍乾燥濃縮、 TCA 濃縮及逆向 HPLC 分析 ULS 上清液之蛋白及小分子 27
C. 以液相-液相分配分析 ULS 上清液小分子成分 28
D. 正向 HPLC 自二次EA 層分離抗發炎及抗癌活性分子 29
討論 65
附錄一 70
參考文獻 78

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