【作者】 罗福文；

【导师】 贾玉杰；

【作者基本信息】 大连医科大学 ， 病理学及病理生理学， 2009， 博士

【摘要】 背景:甲壳素是昆虫和甲壳类动物的外壳中所含的一种物质,是由乙酰氨基葡萄糖组成的聚糖。甲壳素经脱乙酰基处理得到壳聚糖,再经过进一步降解,形成壳寡糖。壳寡糖的化学名为聚氨基葡萄糖胺。据研究,壳寡糖是目前已知自然界中唯一带正电荷的碱性多糖,也是唯一的动物性膳食纤维素。由于壳寡糖分子量低,水溶性好,生物活性高,人体吸收好,从而克服了甲壳素不溶于水、不易被人体吸收的技术难题。所以壳寡糖成为当今国内外研究、开发的重点领域。研究表明,壳寡糖具有广泛的生物学活性:提高机体免疫力,抗菌抑菌作用,促进钙及其它矿物质的吸收,增殖双歧杆菌、乳酸菌等人体有益菌群,对降血糖、降血脂、降血压、保护肝功能等有辅助治疗作用。此外,壳寡糖具有明显的抗肿瘤作用,其对肝癌、肺癌、结肠癌、宫颈癌、白血病、肉瘤等都有显著抑制效果,然而壳寡糖抗肿瘤机制十分复杂,是多方面、多层次的过程。肝癌,是全球最为常见的恶性肿瘤之一。因其恶性度高、病情进展快、治疗难度大、生存期短,一般发病后生存时间仅为6个月,故肝癌有癌中之王之称。并且肝癌是富含血管的肿瘤,极易发生转移。目前,临床上的治疗一般以手术、放疗、化疗为三大支柱,但由于肝癌起病隐匿,发展变化快,患者就诊时往往已进入中晚期,失去了最佳手术时机。大多数化疗药物虽可抑制肿瘤细胞过度增生,但毒副作用大,对正常组织细胞也有很强的杀伤作用。故寻找新的抗肝癌药物迫在眉睫。本实验组前期致力于壳寡糖吸收分布状况的研究,发现壳寡糖能被小肠吸收入血。为了揭示壳寡糖抑制肝癌细胞增殖并促进其凋亡的机制,本实验组通过使用荧光物质标记壳寡糖来研究其对肿瘤细胞的作用,进而探讨在肿瘤细胞表面是否存在与壳寡糖特异性结合的受体。再通过检测一些与细胞凋亡相关的蛋白以及与肿瘤转移相关的细胞因子进一步探寻壳寡糖抗肿瘤机制。目的:1、研究壳寡糖在体内的吸收状况以及吸收入血和直接排出的比例。2、通过FITC荧光标记壳寡糖来检测肝癌SMMC-7721细胞表面是否存在与壳寡糖结合的受体。3、检测壳寡糖对人肝癌SMMC-7721细胞的抑制效果及对血管内皮生长因子(VEGF)表达的影响。4、研究壳寡糖能否促进人肝癌SMMC-7721细胞的凋亡以及对凋亡调控蛋白Bcl-2和Caspase-3的影响。方法:1、利用异硫氰酸荧光素FITC标记壳寡糖,并通过聚丙烯酰胺凝胶层析柱(P2凝胶)将游离的FITC除去,再给禁食后的小鼠用FITC标记的壳寡糖灌胃,1小时后取血清和肠溶物,经处理后,通过荧光分光光度计分别检测血清样品和肠内溶物中荧光的强度,进而确定壳寡糖是否被吸收入血,以及入血和直接从肠道排出的比例。2、将FITC标记的壳寡糖单独作用于人肝癌SMMC-7721细胞,并与先用未标记的壳寡糖作用于肝癌细胞再用标记的壳寡糖作用细胞进行比较,观察壳寡糖与肝癌细胞结合是否有饱和现象进而探索肝癌SMMC-7721细胞表面是否存在与壳寡糖特异性结合的受体。3、利用MTT法检测不同浓度壳寡糖作用于人肝癌SMMC-7721细胞不同时间后细胞增殖活性;应用免疫细胞化学和Western-blotting检测壳寡糖作用肝癌细胞后VEGF表达量的变化。4、镜下观察不同浓度壳寡糖作用肝癌细胞后发生的形态学变化,并利用荧光Hoechst 33258染色法检测细胞凋亡状况,通过免疫细胞化学方法研究壳寡糖对肝癌细胞SMMC-7721中Bcl-2和Caspase-3表达的影响。结果:1、FITC能够标记壳寡糖,并且利用P2聚丙烯酰胺凝胶层析柱可以很好的分离出游离的FITC;用标记的壳寡糖给小鼠灌胃,从血清和肠溶物中都检测到荧光强度,两者比值为2:1。2、单独使用标记的壳寡糖作用肝癌细胞在荧光显微镜下观察所有细胞均发出很强的荧光,而先用未标记的壳寡糖作用肝癌细胞再用标记的壳寡糖作用后荧光强度明显减弱。3、壳寡糖对SMMC-7721细胞增殖有明显的抑制作用,呈剂量依赖关系,并且壳寡糖能明显降低SMMC-7721细胞中VEGF的表达。4、壳寡糖能促进SMMC-7721细胞凋亡,并能上调促凋亡蛋白Caspase-3的表达和降低抑制凋亡蛋白Bcl-2的表达。结论:1、壳寡糖能通过小肠被人体吸收,吸收入血的壳寡糖与直接排出的壳寡糖的比例为2:1。2、壳寡糖能与肝癌细胞结合并呈现出饱和性,此现象说明在肝癌细胞表面有可能存在壳寡糖的特异性受体。3、壳寡糖对人肝癌SMMC-7721细胞的增殖有抑制作用,此作用可能是通过抑制VEGF的表达来实现的。4、壳寡糖能促进人肝癌SMMC-7721细胞的凋亡,此作用可能是通过促进Caspase-3的表达和抑制Bcl-2的表达来实现的。更多还原

【Abstract】 Background: Chitin is a substance in the shell of insects and crustaceans. It is one kind of polysaccharide composed of acetyl glucose- mine. Chitin is deacetylated to form chitosan, and be further hydrolyzed to form chitooligosaccharides(COS). The chemical name of COS is poly amino glucosamine. According to survey, COS is the only alkaline positively charged polysaccharide in the known word, and also the only animal dietary cellulose. Because of the low molecular weight, water soluble, high biological activities, well absorbed by the body, thereby COS overcame the technical problems which the chitin does not dissolve in water and can not easily be absorbed into the body. So the researches focus on the development and utilization of COS. Studies have shown that COS has a wide range of biological activities: including increasing the body immunity, anti-bacterial function, promoting the absorption of calcium and other mineral substance, the proliferation of Bifidobacterium, Lactobacillus and other Probiotics, controlling normal serum glucose, lipid and blood pressure, protecting liver function. In addition, COS also has many obvious anti-tumor effects, and especially has a significant inhibitory effect on liver cancer, lung cancer, colon cancer, cervical cancer, leukemia and sarcoma. However, the anti-tumor mechanisms of COS are very complex, which may be multi-faceted, multi-level processes.Liver cancer is one of the global most common malignant tumors. Because of its high malignancy, liver cancer progresses quickly, the treatment is difficult and the patient’s lifetime is limited, and what is more , after morbidity, the patient’s life generally lasts no more than 6 months. Liver cancer has the name of the king among the all kinds of cancer. Liver cancer is extremely easy to metastasize because it is rich in blood vessels. At present, surgery, radiotherapy and chemotherapy are the three main backbones in the clinical treatments for liver cancer. However, because the incidence of liver cancer was not obvious and the course progresses quickly, it always has entered the middle and late stages when the diagnosis has be made, which meas most of the best surgery opportunites have been lost. Although most chemotherapy medicines suppress the excessive prolifer- ation of tumor cells, the poisonous side effects are fairly strong. They also have strong damaging effects to the normal tissue. Therefore seeking for the new anti-liver cancer medicine is in current focus.Our experimental group has engaged in researching on the absorption and distribution of COS in the small intestine. We discovered COS can be absorbed by the small intestine. In order to reveal the mechanism of COS inhibiting liver cells proliferation and promoting apoptosis, our experimental group utilized Fluorescein isothiocyanate(FITC) to label COS to examine its role in the process of anti-tumor, and to explore the existence of COS receptor on the surface of tumor cells. Some apoptosis-related proteins and tumor metastasis-related cytokines were detected to investigate the mechanisms of COS against tumor. This will lay the theoretical basis for developing COS as pharmaceutical ingredients for the treatment of liver cancer.Objective:1.To investigate the absorption of COS in the body, as well as the proportion between the absorption into the blood and direct excretion. 2. FITC was used to label COS in order to detect the existence of the COS binding receptor on the surface of hepatocelular carcinoma SMMC-7721 cell. 3. To research the inhibitory function of COS on human hepatocelular carcinoma cell line SMMC-7721 and determine the effect of COS on the expression of VEGF in the hepatoma cell line. 4. To detect the effects of COS on inducing the apoptosis of human hepatoma SMMC-7721 cell and promoting the express of apoptosis regulatory proteins like Bcl-2 and Caspase-3.[0]Methods: 1. COS labelled with FITCwas passed through the polyacrylamide gel chromatography column (P2 gel) in order to remove the free FITC. The blood and intestinal contents were obtained from the mice treated with FITC-COS via gastric infusion an hour later. The samples′fluorescence intensity was detected by fluorescence spectrophotometer to determine the proportion between the absorption of COS into the blood and direct excretion. 2. In order to detect the existence of COS receptor, on the one side, FITC-COS was used alone to treat human hepatocellular carcinoma SMMC-7721 cells; and on the other side, the unmarked COS was utilized to treat SMMC-7721 cells firstly, then the FITC-COS was used. Fluorencence intensity was compared between the two groups to observe whether there is a saturation when COS combines with the hepatocellular carcinoma cells and investigate the existence of the COS binding receptor on the surface of hepatocellular carcinoma SMMC-7721 cells. 3. MTT assay was applied to evaluate the proliferation of SMMC-7721 cells treated by different concentrations of COS. Immunocytochemistry and Western- blotting were utilized to measure the expression of vascular endothelial growth factor (VEGF) in the SMMC-7721 cells treated by oligochitosan. 4. To observe the morphological changes of SMMC-7721 cells treated by COS of different concentrations, Hoechst 33258 fluorescence staining was adopted to detect cell apoptosis and immunocytochemistry was utilized to measure the expression of Caspase-3 and Bcl-2 in the SMMC-7721 cells treated by COS.Results: 1.FITC can label COS, and the free FITC can be separated effectively by Polyacrylamide gel chromatography column (P2 gel). Samples of both the blood and intestinal contents from the mice with gastric infusion of FITC-COS had fluorescence intensity. The ratio between them is 2:1. 2. The fluorescence intensity of hepatocelular carcinoma cells treated by FITC-COS alone was extremely strong. However, when the SMMC-7721 cells were treated firstly by unmarked COS and then FITC-COS, the fluorescence intensity was obviously weaken. 3. COS significantly inhibited the SMMC-7721 cells proliferating, and the inhibition was in a dose- dependent pattern. The expression of VEGF was lower in COS treated group than that in the control group. 4. COS can accelerate cell apoptosis. The expression of Caspase-3 and Bcl-2 were respectively higher and lower in COS treated group than those in the control group.Conclusion: 1. COS can be absorbed into the blood through the small intestine, the ratio between absorption into the blood and direct excretion is 2:1. 2. COS can combine with the hepatoma cells and show a phenomenon of saturation , which indicates that there might exist COS receptor on the surface of human hepatocellular carcinoma SMMC-7721 cells. 3. COS could down-regulate the expression of VEGF in SMMC-7721 cells. This might be a part molecular mechanism of COS inhibiting the proliferation of SMMC-7721 cells. 4. COS can promote human hepatoma cell apoptosis, this effect may be realized through the promotion of Caspase-3 expression and inhibition of Bcl-2expression. The mechanisms of COS promoting human hepatoma cell apoptosis were possibly that COS up-regulated the expression of Caspase-3 and down-regulated the expression of Bcl-2.更多还原