【作者】 薛美兰；

【导师】 葛银林；

【作者基本信息】 青岛大学 ， 病原生物学， 2012， 博士

【摘要】 背景与目的：褐藻糖胶(fucoidan)是从海带中提取纯化的一种多糖,其主要组成为褐藻糖、硫酸根,以及少量糖醛酸、半乳糖、木糖。褐藻糖胶具有抗肿瘤、抗病毒、抗凝血、降血脂、降血糖、增强免疫力等生物学功能,使之成为天然海洋药物研究的热点。研究证实,褐藻糖胶对肠癌、乳腺癌、肝癌和白血病等均有明显的抑制作用,并能诱导细胞凋亡,而对正常细胞无毒副作用,但对其具体的作用机制尚未阐明。本研究以小鼠乳腺癌细胞4T1为靶细胞,采用体外培养细胞(体外实验)及建立荷瘤小鼠模型(体内实验)方法从分子水平、细胞水平及动物整体水平探讨褐藻糖胶的抗肿瘤活性及其作用机制,为最终将该药开发成为一种新型的天然抗癌药物奠定基础。方法：1.体外实验：体外培养小鼠乳腺癌细胞4T1,采用MTT比色法、荧光染色、流式细胞术(flow cytometry, FCM)、RT-PCR、Western blotting等方法,观察不同浓度褐藻糖胶对4T1细胞增殖和凋亡的影响,探讨褐藻糖胶体外的抗肿瘤活性及其作用机制。2.体内实验：培养4T1细胞接种Balb/c小鼠建立荷瘤小鼠模型,观察褐藻糖胶对荷瘤小鼠移植瘤生长的抑制情况,TUNEL染色检测瘤组织凋亡细胞,Western blot及免疫组化等方法检测褐藻糖胶处理后瘤组织内Bcl-2/Bax、生存素(Survivin)、β连环蛋白(B-catinin)、细胞周期蛋白Cyclin-D1和细胞周期蛋白依赖性蛋白激酶CDK4以及血管内皮生长因子(VEGF)等相关蛋白的表达,探讨褐藻糖胶体内的抗肿瘤活性及其作用机制。结果：1.体外实验：褐藻糖胶(0、50、100、200μg/ml)作用4T1细胞48 h后,在荧光显微镜下观察到褐藻糖胶处理组细胞出现典型的凋亡形态学改变：细胞核固缩、有DNA碎片；RT-PCR及Western blot结果显示,随褐藻糖胶浓度的增加,Bcl-2 mRNA和蛋白表达均下降、Bax mRNA和蛋白表达升高,导致Bcl-2/Bax比值下降；凋亡抑制蛋白Survivin表达增加；线粒体膜电位下降,细胞色素C由线粒体向细胞浆释放增加,Caspase-3的活化率逐渐增加,明显高于对照组(P<0.05);MTT比色结果显示褐藻糖胶能抑制4T1细胞增殖；细胞周期分析表明,经50、100、200μg/ml褐藻糖胶处理48h后,G1期4T1细胞数明显增加,呈明显的G1期阻滞；Western blot结果显示随褐藻糖胶浓度增加,β-catinin、CyclinD1和细胞周期依赖激酶CDK4表达逐渐减少(P<0.05)。另外,褐藻糖胶处理后,VEGF表达也出现减少(P<0.05)。2.体内实验：结果显示褐藻糖胶能抑制荷瘤小鼠移植瘤的增长；TUNEL染色显示褐藻糖胶明显促进肿瘤组织内细胞凋亡；Western blot结果显示Bcl-2/Bax比值降低和Survivin的表达下降；免疫组化检测到β-catinin、CyclinD1、CDK4和VEGF的表达减少,肿瘤组织微血管密度降低；癌灶在肺部的转移率下降。结论：褐藻糖胶能明显诱导4T1细胞凋亡和细胞周期G1期阻滞,抑制小鼠肿瘤生长和肺转移,这些作用可能与Bcl-2/Bax降低、Survivin表达下降、Caspase-3激活以及下调Wnt通路中β-catinin、CyclinD1、CDK4的表达和抑制VEGF的生成有关。更多还原

【Abstract】 BACKGROUND & OBJECTIVE:Fucoidan is a sulfated polysaccharide derived from brown algae that has been reported to perform multiple biological activities, including antitumor activity. Fucoidan could inhibit the growth of tumors, but the mechanism remains unclear. This study aimed to explore the antitumor effects and the related mechanism of fucoidan in vitro and in vivo with mouse breast cancer cell line 4T1 as target cell.METHODS:In vitro, fluorescent staining, flow cytometry, RT-PCR and Western blot were performed to analyze the cell cycle and apoptosis of mouse breast cancer 4T1 cells after treatment of fucoidan at different concentrations (0,50,100,200μg/ml). In vivo, therapy experiments were conducted on Babl/c mice bearing breast cancer. The tumor volume and weight were measured. The number of apoptotic cells in tumor tissues was assessed by TUNEL immunostaining. Western blot were used to detect the expression of Bcl-2, Bax, and Survivin in excised tumors. Immunohistochemical assays were used to detect the expression of Wnt/β-catenin signaling agents in tissues.RESULTS:When treated with fucoidan for 48h,4T1 cells showed typical apoptotic morphologic changes including chromatin condensation and DNA fragment. Along with the increase of fucoidan concentration, Bcl-2 expression was decreased, Bax expression was increased, and the ratio of Bcl-2 to Bax was significantly decreased (P<0.05). The expression of Survivin in 4T1 Cells was decreased when treated with fucoidan (P<0.05). Mitochondrial transmembrane potential was decreased and cytosyl cyt-C proteins increased in 4T1 cells after 48 h treatment of fucoidan detected by Western blot. When treated with fucoidan (50、100,200μg/ml) for 48 h, the activated Caspase-3 in 4T1 cells were significantly higher than that in control 4T1 cells (P<0.05).Moreover, the expression of VEGF was decreased when treated with fucoidan. In addition, the proportion of 4T1 cells at G1 phase was significantly increased with an apparent G1 phase arrest when treated with fucoidan (50,100,200μg/ml) for 48 h. In the progress of cell cycle arrest induced by fucoidan, the expression of B-catenin, cyclin-D1 and phosphorylated CDK4 were down-regulated (P<0.05).Intraperitoneal injection of fucoidan in subcutaneous breast cancer models reduced the tumor volume, induced apoptosis, inhibited angiogenesis in tumor tissue and decreased pulmonary metastases.CONCLUSION:Fucoidan could induce apoptosis and G1 phase arrest in 4T1 cells, possibly by decreasing the expression of Bcl-2/Bax and Survivin, activating Caspase-3, down-regulating the expression of B-catenin, cyclin-D1 and phosphorylated CDK4. Fucoidan also could inhibit tumor angiogenesis by down-regulating the expression of VEGF. These findings indicate that crude fucoidans inhibited mouse breast cancer growth in vitro and in vivo.These data suggest that fucoidan may serve as a potential therapeutic agent for breast cancer.更多还原