Study on the inhibition of human carcinoma cells by the brown alga Petalonia binghamiae (Phaeophyceae) extracts

本論文是從海洋褐藻小海帶萃取多種多醣類與蛋白質，經過分離純化後對人 類肝癌細胞株Hep G2、人類胰臟癌細胞株MIA PaCa-2及人類乳癌細胞株 MDA-MB-231做體外活性檢測。結果顯示小海帶Fucoidan、褐藻酸、褐藻酸鈣及 褐藻酸鈉等多醣，均能顯著抑制癌細胞生長。褐藻酸鈉的抑癌活性在經過檸檬酸 水解後顯著地降低，而遇到鈣，鋅，鈷，錳等二價離子則會失去抑制活性。但， 這些金屬離子若經過Na2EDTA的前處理就不會影響褐藻酸鈉的抑癌活性。上述 結果顯示在褐藻酸羥基上的離子交換情形以及分子構形是影響該多糖抑制癌細 胞生長反應的重要因素。再者，褐藻酸鈉能促進老鼠巨噬細胞增殖及分泌蛋白質 類物質。分泌的蛋白質能顯著抑制癌細胞的生長。小海帶以PBS萃取，並經硫酸 銨沈澱、膠體過濾管柱與離子交換管柱之層析及純化後，所得之小海帶活性之蛋 白質（簡稱 EBP），其蛋白質的分子量為69 kDa，醣含量則佔20.37 %，屬一種 醣蛋白，亦為單聚體蛋白。此醣蛋白對上述的癌細胞有顯著抑制增殖的效果。 EBP對酸鹼值的變化亦具耐受性，在pH值3.0～12.0範圍內均能維持很高的活性。 再者，EBP於121℃下加熱20分鐘，仍保有原來活性的53%，顯示EBP也具耐熱性。 由本實驗得知小海帶具有抑制癌細胞生長活性之醣蛋白與多醣類，值得進一步研究，以期未來可被應用在醫藥上。Various extracts of the marine brown alga Endarachne binghamiae were assayed in vitro for inhibiting the proliferation in human breast adenocarcinoma cell （MDA-MB-231）, human hepatoblastoma cell（Hep G2）and human pancreatic cell （MIA PaCa-2）. In the present study, fucoidan, alginic acid, calcium alginate and sodium alginate extracted from the brown alga Petalonia binghamiae inhibited markedly the proliferation in carcinoma cells. The inhibitory reaction of sodium alginate decreased after being treated with citrate buffer solution and lost in the presence of Ca2+, Zn2+, Co2+ or Mn2+. However, sodium alginate remained high inhibitory activity if metal ions were treated with Na2EDTA prior to addition to culture medium. These results showed that ion exchange at carboxyl groups as well as molecule conformation of the polymer played an important role in alginate inhibitory reaction with carcinoma cells. Moreover, the obtained sodium alginate induced murine macrophages to produce proteinaeous substance that significantly inhibited carcinoma cell proliferation. My study suggests that sodium alginate has using potential in anticancer therapy and research due to its wide distribution and high production in marine brown algae. A novel protein assigned as EBP (Endarachne binghamiae protein) was obtained after being extracted in phosphate buffered saline, precipitated by saturated ammonium sulfate and finally purified by gel filtration and ion-exchange column chromatography. The purified protein has molecular weight of 69 kDa. It is a monomer glycoprotein, containing 20.37 % carbohydrate moiety. The EBP is rather stable in immunity while subjected to 121℃ and a wide range of pH values (3.0~12.0). The above findings suggests that the reported alga is a promising source of proteins and polysaccharides. In detail research, we are looking forward to seeing that immunostimulation and immuno-modulation can be used as medicine in cancer therapy.目錄 中文摘要………………………………………………………………………… I 英文摘要…………………………………………………………………………. II 第一章緒論………………………………………………………………….. 1 第二章材料與方法………………………………………………………… 7 一、實驗材料……………………………………………………………… 7 二、實驗方法……………………………………………………………… 14 第三章結果………………………………………………………………….. 31 第四章討論………………………………………………………………….. 46 參考文獻…………………………………………………………………….…… 108 附錄………………………………………………………………………………. 12