幾丁寡醣（Chitosan oligosaccharides）廣泛存在於甲殼類動物的外骨骼、真菌類、昆蟲與酵母菌的細胞壁中，為幾丁質與幾丁聚醣進一步以化學或酵素法分解而成之無毒性小分子，並且具有低黏度、低分子量及水溶性等特性。由於過去的研究指出幾丁寡醣具有降低膽固醇、抗氧化、增強免疫活性、抗菌及抗腫瘤生長的功能，近年來幾丁聚醣及幾丁寡醣已經成為熱門的健康食品。但由於幾丁寡醣在抗腫瘤生長與抗惡性癌細胞轉移相關機轉的資料目前仍缺乏並有待於進一步的研究。本研究目的為評估由真菌中萃取之真菌幾丁寡醣之抗癌及抗轉移能力及其相關機制之探討。在體外（in vitro）實驗方面，利用MTT assay方法篩選真菌幾丁寡醣對不同癌細胞株抑制生長情形，結果顯示真菌幾丁寡醣抑制人類肝癌細胞（HepG2 cell）生長效果最顯著（抑制能力約達50 %），另外真菌幾丁寡醣於細胞週期中可以抑制S phase，進一步利用BrdU cell cycle analysis assay分析細胞DNA合成之情形，發現HepG2 細胞加入真菌幾丁寡醣後S phase有被抑制之現象，而於西方墨點法分析細胞週期相關蛋白中，cyclin D3、p21及p27表現有上升的趨勢而cyclin A、cyclin B及cdk-2有下降的趨勢；而跟癌症轉移相關的蛋白之ㄧ（MMP-9），其活性也有顯著之下降。在體內（in vivo）實驗方面，在SCID mice抗腫瘤生長動物模式中，高劑量真菌幾丁寡醣略有抑制人類肝癌腫瘤生長之情形。另外在真菌幾丁寡醣抗轉移模式中，利用LLC-bearing mice腫瘤生長與肺轉移（切除或不切除腫瘤）這兩種模式來評估真菌幾丁寡醣是否具有抗癌症轉移之能力，不論是切除或不切除腫瘤模式中，隨著劑量之上升，初期原發性腫瘤體積及肺轉移之程度有下降趨勢。結果顯示，真菌幾丁寡醣可能於癌症之化學預防具有其潛在抗癌之能力。

　　Chitosan oligosaccharides are widely present in the exoskeleton of crustaceans and in cell walls of fungi, insects and yeast. There are nontoxic oligomers derived from chitin and chitosan by either chemical or enzymatic hydrolysis. Previous studies indicated that chitosan oligosaccharides have ability to lower hyperlipidaemia, antioxidant, immuno-enhancing effects, antifungal and anti-tumor activity, making it becoming a popular healthful food in the past few years. So far, the mechanisms of anti-tumor growth and anti-metastasis of malignant cells by chitosan oligosaccharides are unclear and needed further investigation. In the present works, the in vitro and in vivo studies were designed to evaluate the anti-cancer potency and related pathways of chitosan oligosaccharides. In in vitro studies, we found that chitosan oligosaccharides significantly inhibited human hepatocellular carcinoma (HepG2 cells) proliferation by using MTT assay. Flow cytometry analysis of cell cycle distribution indicated that the percentage of S phase reduced in cells treated with chitosan oligosaccharides. In addition, BrdU incorporation assay revealed a decreased DNA synthesis rate in chitosan-treated HepG2 cells. Cell cycle-related gene expression were analyzed by western blot and the results indicated that cyclin D3, p21 and p27 were up-regulated, while the cyclin A, cyclin B and cdk-2 were down-regulated. Moreover, we also found that the activity of one of metastasis related proteins (MMP-9) could be inhibited in LLC cells . In in vivo studies, we also found that chitosan oligosaccharides inhibited the growth of HepG2 xenografts in SCID mice. In LLC-bearing mice tumor growth and lung metastasis model, tumor volume in early stage and lung colonies could be inhibited by chitosan oligosaccharides. Our results suggest a potential anti-cancer potency of chitosan oligosaccharides in caner chemoprevention.

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