臺灣博碩士論文加值系統

蜂膠(Propolis)是由蜜蜂蜂巢所得來的，東方國家將蜂膠當作民間醫療使用。本研究採用市售蜂膠乙醇萃取物(ethanol extract of propolis, EEP)及其活性成分Caffeic acid phenethyl ester (CAPE)進行生體外免疫調節功能活性之評估。EEP和CAPE各別在0.25 mg及3.0 g濃度下具有抑制人類子宮頸癌細胞株 (HeLa cells)生長效果；EEP及CAPE均能降低白血病細胞株 (Leukemia cell lines)如HL-60、U-937的存活率。EEP於0.5 mg/mL，CAPE於濃度6.25 g/mL均顯現癌細胞的DNA片段化，此抑制效果於細胞上出現皺縮及凋亡體之形態學特徵上的改變。以10 mM TPA活化人類單核球細胞株(THP-1)分化為巨噬細胞，CAPE於10 g/mL濃度時，THP-1細胞株吞噬E. coli bacteria 的能力為7.66%，增加至20 g/mL時，降為4.05%，顯示CAPE可以抑制TPA所誘導之發炎狀態，故可降低THP-1之吞噬活性。小鼠脾臟細胞當加入CAPE 12.5 g劑量時，可增加殺死小鼠淋巴癌細胞株(YAC-1)的數目，增加自然殺手細胞(NK cells)活性能力達3.42%，並隨著劑量的增加，NK活性也愈大；EEP 0.5 mg濃度可增加55.44% NK活性，顯示Propolis能增強小鼠免疫力。EEP作用於老鼠巨噬細胞 J774.1，在濃度0.25 mg/mL下，具有抑制LPS 1 g/mL併用IFN-γ10 U/mL所誘導之NO釋出量；對另一株老鼠巨噬細胞RAW264.7，於濃度0.5 mg/mL時亦具有抑制NO的釋出。CAPE於濃度6.25 g/mL即有抑制老鼠巨噬細胞株J774.1之iNOS蛋白催化活性，當濃度高達25 g/mL則達到完全抑制效果。此外，以TPA誘導人類乳癌細胞株(MCF-7、MDA-MB-453)產生之COX-2，CAPE 於20 g/mL劑量亦具有抑制COX-2蛋白表現作用。進一步以RT-PCR確認J774.1細胞在EEP濃度6.25 g/mL，可完全抑制其基因的表現。而CAPE於濃度1.25M時亦可抑制iNOS基因表現。另以LPS併用IFN-γ誘導老鼠巨噬細胞株J774.1，應用生物晶片做成cDNA微矩陣( microarry )研究，分析其基因表現情形，結果顯示J774.1細胞株有510個基因有表現，285個基因上調( upregulatory )，225個基因下調( downregulatory )。此外，以TPA誘導人類乳癌細胞株( MCF-7 )，使用8000個人類cDNA微矩陣雜交，結果顯示MCF-7細胞株有279個基因有表現，215個基因上調，64個基因下調。這些cDNA微矩陣結合生物資訊 (bioinfor-matics ) 能對調節基因之重要性及影響層面，提供更深一層的訊息。

Propolis obtained from honeybee hives has been used in Oriental folk medicine. Caffeic acid phenethyl ester (CAPE) is an active ingredient isolated from propolis. The present study, assessment the effect of ethanol extract of propolis (EEP) and CAPE that possesses anti-cancer, anti-inflammatory, and immunomodulatory activity. EEP was analysed for the determination of total flavonoid content by using the aluminium nitrate method, UV spectrophotometry. EEP contained high total flavonoid content. It was observed EEP showed possessed stronger DPPH free radical scavenging activity. We was found that EEP and CAPE entered leukemia cells and then inhibited their survival in a concentration-dependent manner. EEP induced characteristic DNA fragmentation and morphological changes typical of apoptosis in these cells. These results suggest that EEP is a potent apoptosis-inducing agent in human leukemia cells. EEP and CAPE signifycantly inhibited NO production and iNOS protein expression induced by lipopolysaccharide (LPS) plussing interferon gamma. EEP and CAPE also signifycantly inhibited COX-2 protein expression induced by Phorbol myristate acetate (TPA). These results suggest that EEP and CAPE may exert its anti-inflammatory effect by inhibiting the iNOS gene expression and by directly inhibiting the protein activity of iNOS and COX-2. Finally, analysis of gene expression at the RNA level using cDNA against an array of 15000 mice genes showed that 510 (3.4%) of the genes were expressed in the J774.1 cells. A total of 285 genes (1.9 %) were found to be upregulated and 225 genes (1.5%) were found to be downregulated. Analysis of gene expression at the RNA level using cDNA against an array of 8000 human genes showed that 279 (3.5%) of the genes were expressed in the MCF-7 cells. A total of 215 genes (2.7%) were found to be upregulated and 64 genes (0.8%) were found to be downregulated. This using of cDNA microarry coupled to bioinformations can provide in-depth information on the impact and importance of the regulated genes.

中文摘要 I
英文摘要…………………………………………………………...……II
壹、 前言 1
貳、 文獻整理 2
一、蜂膠(Propolis) 2
二、蜂膠加工處理過程 3
三、蜂膠之組成………………………………………………… 3
四、蜂膠之用途………………………………………………… 4
五、蜂膠之用量與LD50………………………………………….. 5
六、蜂膠之毒性 6
七、蜂膠的生理和藥理特性 6
1. 抗發炎作用(Anti-inflammatory effect) 6
2. 抗菌性(Antibacterial activity) 7
3. 抗病毒性(Antiviral activity) 8
4. 抗腫瘤效果(Antitumour effect)及抗突變作用(Antimutagenesis) 9
5. 免疫活化作用 10
6. 抗氧化作用(Antioxidative effect) 11
7. 保肝效果(Hepato-protective effect) 12
八、氧自由基與疾病…………………………………………… 12
九、氧化氮(Nitric Oxide, NO)，氧化氮合成酶(NOS)和
可誘導型氧化氮合成酶(iNOS) 13
十、前列腺素(Prostaglandins, PGs)和第二型環氧酶(Cyclooxygenase-2, COX-2) 14
十一、PGS、COX-2 表現與癌化反應 15
十二、免疫功能評估 16
1. 自然殺手細胞之活性 16
2. 吞噬細胞之活性 17
3. 淋巴球活化 17
十三、衛生署免疫調節功能評估方法………………………...18
十四、生物晶片(Biochip) 18
參、 材料與方法 24
一、 實驗大綱 24
二、 實驗材料 25
1. 市售蜂膠來源 25
2. 細胞株 25
3. 藥品 25
4. 設備 29
三、 實驗項目與實驗方法 31
1. 市售蜂膠萃取液之篩選 31
2. 細胞培養 32
3. 細胞存活率(MTT Assay) 33
4. DNA片段化測定……………………………..………..33
5. 吞噬細胞活性分析 34
6. 自然殺手細胞活性分析………………………………35
7. NO釋放測定 36
8. Immunoblot 分析－iNOS Activity 37
9. RT-PCR分析－iNOS Gene Expression 41
10. Immunoblot 分析－COX-2 Activity 44
11.生物晶片分析 45
12.統計方法 46
肆、 結果與討論 47
一、 市售蜂膠萃取液之篩選 47
1. 蜂膠萃取液之抗氧化試驗(Antioxidative Activity) 47
2. 蜂膠萃取液之HPLC測定(HPLC Separation) 47
3. 蜂膠萃取液之還原力測定(Reducing Power) 47
4. 蜂膠萃取液之類黃酮總量測定(Total Flavonoid Determination) 47
二、 細胞存活率 48
三、 DNA片段化 48
四、吞噬細胞活性分析 49
五、自然殺手細胞活性分析 49
六、NO釋放量 50
七、Immunoblot 分析－iNOS Activity 51
八、RT-PCR分析－iNOS Gene Expression 51
九、Immunoblot 分析－COX-2 Activity 51
十、 生物晶片分析 52
1. 老鼠巨噬細胞株J774.1之微矩陣基因表現分析 52
2. 人類乳癌細胞株MCF-7之微矩陣基因表現分析…52
伍、結論 54
陸、參考文獻 55
柒、圖表 66

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