近年來，隨著健康意識抬頭，空氣污染的問題逐漸受到重視，環境中PM2對公眾健康造成的影響已經成為全球人們關注的焦點，近期多個研究指出，肺癌的發生率提高和PM2有著密不可分的關係。在台灣，癌症一直是十大死因之首，且以肺癌的致死率最高，這可能與空氣汙染中的PM2有關。現代社會，人們越來越注重養生，從天然來源萃取出的多醣日益受到關注，台灣四面環海，海洋資源豐富，本研究所使用的海木耳屬於紅藻的一種，利用熱水萃取的方式萃得海木耳水萃物，實驗動物為C57BL/6，將小鼠進行分組後，腫瘤背負組皮下注射小鼠肺癌細胞 (LLC)，再以噴霧的方式讓各兩的組正常小鼠和腫瘤背負小鼠暴露於PM2的環境中，且其中一組餵食海木耳多醣，探討海木耳水萃物抗腫瘤的效果，結果顯示，海木耳水萃物具有抗氧化及抗發炎的能力，在細胞實驗中也觀察到其具有抑制癌細胞生長、遷移和侵襲的能力，動物實驗結果顯示，海木耳水萃物可輔助改善腫瘤所引起的惡病質症狀，也能抑制腫瘤的轉移及生長。後續分析發現海木耳水萃物會增加毒殺型 T 細胞 (Cytotoxic T cell) 及自然殺手細胞 (NK cell) 表現亮，經 ELISA 測定小鼠血清中 IFN-γ 表現量，發現餵食海木耳水萃物能使小鼠的 IFN-γ 表現量上升，因此推測海木耳水萃物是藉由活化 Th1 路徑來達到毒殺癌細胞的效果。並提升免疫細胞數量，達到毒殺癌細胞之效果。

Growing health consciousness in recent years has led people to pay more attention to air pollution, The impacts of ambient PM2 on public health had become great concerns worldwide. Recently, more and more studies indicate that PM2 has been associated with an increase in lung cancer incidence. In Taiwan, cancer is the first among the top ten leading causes of death, and lung cancer has the highest fatality rate. Taiwan is surrounded by sea, and have abundant ocean sources. Sarcodia montagneana is a kind of red seaweed, in this study we used Sarcodia montagneana water extracts to fed C57BL/6 mice. During the experiment, we use an atomizer to spray PM2, made normal mice and tumor-bearing mice exposed to PM2. The goal of the study is to evaluate the anti-tumor effect of Sarcodia montagneana water extracts. In vitro, Sarcodia montagneana water extracts could inhibit LLC cells on migration and invasion in transwell assay. In vivo, Sarcodia montagneana water extracts could increase the immune cell, decrease body-weight loss and tumor weight, through ELISA kit analysis, we found that the group who have been fed Sarcodia montagneana water extracts, increased the level of IFN-γ in serum. In conclusion, Sarcodia montagneana water extracts could anti-tumor growth, cancer cachexia and inhibit metastatic activities through Th1 pathway.

摘要 I
Abstract II
目錄 III
壹、前言 1
貳、文獻回顧 3
一、癌症 3
二、肺癌 (Lung cancer) 3
1. 肺癌病理學 3
2. 肺癌流行病學 3
3. 肺癌特徵 4
4. 肺癌的治療與困境 4
二、懸浮微粒 (Particulate matter, PM) 4
1. 空氣中的懸浮微粒 4
2. 懸浮微粒與疾病之間的關係 5
三、藻類 5
1. 海藻(Seaweed) 5
1.1海藻之簡介 5
1.2台灣海藻的分布 6
1.3紅藻 (Red algae) 6
2. 海木耳 (Sarcodia montagneana) 6
2.1海木耳之簡介 6
2.2海木耳生理活性 7
參、實驗設計流程 8
一、海木耳萃取及特性分析 8
二、細胞實驗 (In vitro) 8
三、動物實驗 (In vivo ) 9
肆、材料與方法 10
一、實驗材料 10
1. 實驗細胞株 10
2. 細胞培養關試劑 10
3. 海木耳來源 10
4. PM2來源 10
5. 細胞存活率試劑 10
6. 抗氧化試驗 10
7. 動物實驗 11
8. 細胞激素分析 11
9. 蛋白質萃取與西方墨點法相關試劑 11
10. 組織切片 12
11. 免疫螢光染色 12
12. 免疫化學染色 12
二、實驗方法 13
1. 細胞株之保存活化與培養 13
1.1 細胞株之保存 13
1.2 細胞株活化 13
1.3 細胞培養 13
2. 海木耳樣品製備 13
2.1 熱水萃取法 14
3. PM2收集 14
4. 樣品特性分析 14
4.2 總醣含量測定 (Total sugar content) 14
4.3 硫酸根含量測定 15
4.4 海木耳對細胞毒性測試 15
4.5 抗氧化能力試驗 15
4.6 細胞內一氧化氮含量測定 17
5.細胞遷移試驗 17
5.1 傷口癒合試驗 17
5.2細胞遷移和侵襲試驗 17
5.3 細胞侵襲試驗 18
6. 動物實驗 (In vivo) 18
6.1 實驗小鼠模式建立 18
6.2 組織器官分析 18
7. 統計分析 19
伍、結果 20
一、海木耳水萃物特性分析 20
二、海木耳水萃物抗氧化活性之探討 20
三、體外試驗 20
1. 海木耳水萃物對細胞毒性之影響 20
2. PM2對細胞毒性之影響 20
3. 海木耳水萃物對巨噬細胞吞噬能力之影響 20
4. PM2對巨噬細胞吞噬能力之影響 21
5. 傷口癒合試驗 21
6. 細胞遷移及侵襲試驗 21
四、利用動物實驗探討海木耳水萃物及PM2對腫瘤背負小鼠之影響 22
1. 海木耳水萃物及PM2對腫瘤生長之影響 22
2. 海木耳水萃物及PM2對肺癌腫瘤背負小鼠體重變化之影響 22
3. 海木耳水萃物及PM2對肌肉及脂肪之影響 22
4. 海木耳水萃物及PM2對腫瘤組織中 COX-2 蛋白表現量之影響 22
5. 海木耳水萃物抑制腫瘤轉移 22
6. 海木耳水萃物對淋巴細胞之調節作用 23
7. 海木耳水萃物對Th1及Th2之調節作用 23
8. 海木耳水萃物對腫瘤血管新生的影響 23
陸、討論 24
柒、結論 28
捌、參考文獻 29
玖、圖表 36


 

王含羽。2012。牛樟芝-中草藥發酵產物對第二型登革病毒感染之保護效果評估。國立台灣海洋大學食品科學系碩士學位論文。基隆。台灣。
王斌全、宋彬、張剛利、張革紅、周芸、趙和平、賈軍梅、康宏春。2006。腫瘤學概論。軍事醫學科學出版社。北京。
行政院農委會水產試驗所。2007。基隆。台灣。
邱雅凰。2012。探討藻類多醣抗病毒與其免疫調節之作用機制。國立台灣海洋大學食品科學系博士學位論文。基隆。台灣。
財團法人台灣癌症基金會。2019。台北。台灣
國立台灣博物館。2004。台灣海藻資料網。台北。台灣。
張睿昇。2003。海藻世界。臺北：泛亞國際。
陳正平。2005。台灣沿海、綠島及蘭嶼海洋生物多樣性之調查研究。國立台灣中山大學海洋植物研究所博士論文。
衛生福利部。2017。癌症防治。疾病管制署。臺北。臺灣。
衛生福利部。2018。106年國人十大死因。台北。台灣。
Alghamdi, H. I., Alshehri, A. F., & Farhat, G. N. (2018). An overview of mortality & predictors of small-cell and non-small cell lung cancer among Saudi patients. Journal of epidemiology and global health, 7, S1-S6.
Campos, A., Souza, C. B., Lhullier, C., Falkenberg, M., Schenkel, E. P., Ribeiro‐do‐Valle, R. M., & Siqueira, J. M. (2012). Anti‐tumour effects of elatol, a marine derivative compound obtained from red algae Laurencia microcladia. Journal of pharmacy and pharmacology, 64(8), 1146-1154.
Candido, J., & Hagemann, T. (2013). Cancer-related inflammation. Journal of clinical immunology, 33(1), 79-84.
Chen, M. Z., Yu, J., Long, Z. J., & Luo, Q. B. (2005). Studies on antimutagenic and free radical scavenging effect of polysaccharide from gracilaria lemaneiformis. Food science, 26, 219-222.
Deng, Z. F., & Ji, M. H. (1995). Composition of polysaccharides of Gracilaria Sjeostedtii kylin and G. Textorii (SUR.) De-Toni (Rhodophyta) and their antitumor effect. Oceanol et limnol sin, 26, 575-581.
Dinis, T. C. P., Madeira, V. M. C., & Almeida, M. L. M. (1994). Action of phenolic derivates (acetoaminophen, salycilate and 5-aminosalycilate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Archives of biochemistry and biophysics, 315, 161-169.
Dodgson, K. (1961). Determination of inorganic sulphate in studies on the enzymic and non-enzymic hydrolysis of carbohydrate and other sulphate esters. Biochemical journal, 78, 312.
Falcon-Rodriguez, C. I., Osornio-Vargas, A. R., Sada-Ovalle, I., & Segura-Medina, P. (2016). Aeroparticles, composition, and lung diseases. Frontiers in immunology, 7, 3.
Fan, Y., Lin, M., Luo, A., Chun, Z., & Luo, A. (2014). Characterization and antitumor activity of a polysaccharide from Sarcodia ceylonensis. Molecules, 19(8), 10863-10876.
Golstein, P., & Griffiths, G. M. (2018). An early history of T cell-mediated cytotoxicity. Nature reviews immunology, 1.
Gross, A., McDonnell, J. M., & Korsmeyer, S. J. (1999). BCL-2 family members and the mitochondria in apoptosis. Genes & development, 13(15), 1899-1911.
He, J., Xu, Y., Chen, H., & Sun, P. (2016). Extraction, structural characterization, and potential antioxidant activity of the polysaccharides from four seaweeds. International journal of molecular sciences,17(12), 1988.
Hsu, H. Y., Lin, T. Y., Wu, Y. C., Tsao, S. M., Hwang, P. A., Shih, Y. W., & Hsu, J. (2014). Fucoidan inhibition of lung cancer in vivo and in vitro: Role of the Smurf2-dependent ubiquitin proteasome pathway in TGFβ receptor degradation. Oncotarget, 5(17), 7870.
Janeway Jr, C. A., & Medzhitov, R. (2002). Innate immune recognition.Annual review of immunology,20(1), 197-216.
Kang, K. S., Kim, I. D., Kwon, R. H., Lee, J. Y., Kang, J. S., & Ha, B. J. (2008). The effects of fucoidan extracts on CCl 4-induced liver injury. Archives of pharmacal research, 31(5), 622.
Kuda, T., Tsunekawa, M., Goto, H. & Araki, Y. (2005). Antioxidant properties of four edible algae harvested in the Noto Peninsula. Journal of food composition and analysis, 18, 625-633.
Lee, S. H., Ko, C. I., Jee, Y., Jeong, Y., Kim, M., Kim, J. S., & Jeon, Y. J. (2013). Anti-inflammatory effect of fucoidan extracted from Ecklonia cava in zebrafish model. Carbohydrate polymers, 92(1), 84-89.
Lewis, J. L., & Norris, J. N. (1987). A history and annotated account of the benthic marine algae of Taiwan. Smithsonian contributions to the marine sciences.
Lu, X, Yao, T, Fung, JC, and Lin, C (2016). Estimation of health and economic costs of air pollution over the Pearl River Delta region in China. Sci total environ. 566–567, 134-43.
Mantovani, A., Allavena, P., Sica, A., & Balkwill, F. (2008). Cancer-related inflammation. Nature, 454(7203), 436.
McCord, J. M., & Fridovich, I. (1969). The utility of superoxide dismutase in studying free radical reactions I. radicals generated by the interaction of sulfite, dimethyl sulfoxide, and oxygen. Journal of biological chemistry, 244(22), 6056-6063.
McTiernan, A., Jinks, R. C., Sydes, M. R., Uscinska, B., Hook, J. M., van Glabbeke, M., Hogendoorn, P. C. (2012). Presence of chemotherapy-induced toxicity predicts improved survival in patients with localised extremity osteosarcoma treated with doxorubicin and cisplatin: a report from the European Osteosarcoma Intergroup. European journal of cancer, 48(5), 703-712.
Ni, L, Chuang, CC, and Zuo, L (2015). Fine particulate matter in acute exacerbation of COPD. Front physiol. 6, 294.
Oh, S. Y., Heo, J., Noh, O. K., Chun, M., Cho, O., & Oh, Y. T. (2018). Absolute Lymphocyte Count in Preoperative Chemoradiotherapy for Rectal Cancer: Changes Over Time and Prognostic Significance. Technology in cancer research & treatment, 17, 1533033818780065.
Op den Kamp, C. M., Gosker, H. R., Lagarde, S., Tan, D. Y., Snepvangers, F. J., Dingemans, A. M. C., ... & Schols, A. M. (2015). Preserved muscle oxidative metabolic phenotype in newly diagnosed non‐small cell lung cancer cachexia. Journal of cachexia sarcopenia and muscle, 6(2), 164-173.
Robak, J. & Gryglewski, R. J. (1988). Flavonoids are scavengers of superoxide anions. Biochem pharmacol, 37, 837-841.
Shacter, E., & Weitzman, S. A. (2002). Chronic inflammation and cancer. 16(2), 217-229.
Sancini, G., Farina, F., Battaglia, C., Cifola, I., Mangano, E., Mantecca, P., Palestini, P. (2014). Health risk assessment for air pollutants: alterations in lung and cardiac gene expression in mice exposed to Milano winter fine particulate matter (PM2. 5). Plos one, 9(10), e109685.
Shimada, K., Fujikawa, K., Yahara, K. & Nakamura, T. (1992). Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. Journal of Agricultural and food chemistry, 40, 945-948.
Solheim, T. S., Blum, D., Fayers, P. M., Hjermstad, M. J., Stene, G. B., Strasser, F., & Kaasa, S. (2014). Weight loss, appetite loss and food intake in cancer patients with cancer cachexia: three peas in a pod analysis from a multicenter cross sectional study. Acta oncologica, 53(4), 539-546.
Stevanović, I, Jovasević-Stojanović, M, and Stosić, JJ (2016). Association between ambient air pollution, meteorological conditions and exacerbations of asthma and chronic obstructive pulmonary disease in adult citizens of the town of Smederevo. Vojnosanit pregl. 73, 152-8.
Thatcher, N., Chang, A., Parikh, P., Pereira, J. R., Ciuleanu, T., Von Pawel, J., Carroll, K. (2005). Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). The lancet, 366(9496), 1527-1537.
Tsujii, M., Kawano, S., Tsuji, S., Sawaoka, H., Hori, M., DuBois, R. N. (1998). Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell, 93(5), 705-716.
Vinikoor-Imler, L. C., Davis, J. A., & Luben, T. J. (2011). An ecologic analysis of county-level PM2. 5 concentrations and lung cancer incidence and mortality. International journal of environmental research and public health, 8(6), 1865-1871.
Wang, J., Shu, J. Z., Chen, X. Y. (2013). The effect of PM2. 5 carrier components on respiratory health. Chinese journal of tuberculosis and respiratory diseases, 36(12), 970-972.
Wang, J., Zhang, Q., Zhang, Z., Song, H., Li, P. (2010). Potential antioxidant and anticoagulant capacity of low molecular weight fucoidan fractions extracted from Laminaria japonica. International journal of biological macromolecules, 46(1), 6-12.
Weitzman, S. A., & Gordon, L. I. (1990). Inflammation and cancer: role of phagocyte-generated oxidants in carcinogenesis. Blood, 76(4), 655-663.
World Health Organization. (2010). WHO handbook for reporting results of cancer treatment.
Wijesinghe, W. A. J. P., & Jeon, Y. J. (2012). Biological activities and potential industrial applications of fucose rich sulfated polysaccharides and fucoidans isolated from brown seaweeds: A review. Carbohydrate Polymers, 88(1), 13-20.
Yang, B., Chen, D., Zhao, H., Xiao, C. (2016). The effects for PM2. 5 exposure on non-small-cell lung cancer induced motility and proliferation. Springerplus, 5(1), 2059.
Yermak, I. M., Barabanova, A. O., Aminin, D. L., Davydova, V. N., Sokolova, E. V., Solov’eva, T. F., ... & Shin, K. S. (2012). Effects of structural peculiarities of carrageenans on their immunomodulatory and anticoagulant activities. Carbohydrate polymers, 87(1), 713-720.
Zhang, M., Mueller, N. T., Wang, H., Hong, X., Appel, L. J., & Wang, X. (2018). Maternal Exposure to Ambient Particulate Matter≤ 2.5 µm During Pregnancy and the Risk for High Blood Pressure in Childhood. Hypertension, Hypertensionaha,117.
Zhishen, J., Mengcheng, T., Jianming W. (1999). The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food chemisrty, 64, 555-559.
Zhao, H., Yang, B., Xu, J., Chen, D. M., & Xiao, C. L. (2017). PM2. 5-induced alterations of cell cycle associated gene expression in lung cancer cells and rat lung tissues. Environmental toxicology and pharmacology, 52, 77-82.
Zhou, S., Ye, W., & Zhang, M. (2012). The effects of nrf2 on tumor angiogenesis: a review of the possible mechanisms of action. Critical review in eukaryotic gene expression, 22(2).
Zhou, W., Tian, D., He, J., Wang, Y., Zhang, L., & Cui, L. (2016). Repeated PM2. 5 exposure inhibits BEAS-2B cell P53 expression through ROS-Akt-DNMT3B pathway-mediated promoter hypermethylation. Oncotarget, 7(15), 20691.