| 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. |
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