Effect And Mechanism Of Chitooligosaccharide And Its Derivatives On Anti-tumor And Immune Regulation

The ocean accounts for 71% of the earth's surface, in which more than 400,000 organisms live, accounting for 80% of the total number of creatures on earth. These marine organisms live in a unique environment (high salinity, high pressure with lack of oxygen and sunlight), which faciliates the production and accumulation of compounds with special chemical structures and biological functions, many of which can be a vital source for developing new marine medicines and health foods.Chitooligosaccharide (COS), obtained by deacetylation of chitin, and its derivatives demonstrate good biocompatibility and bioactivity, and have recently become research focus.This dissertation performed studies on anti-tumor and immunological activities of COS, and its derivatives including D-glucosamine, N-acetylglucosamine (NAG) by in vivo and in vitro experiments. Cell morphology, flow cytometry, immunochemistry, and Western Blot methods were adopted to systematically explore their functional relations.The results demonstrat that D-glucosamine can inhibit the growth of SGC-7901 cells between the concentration range of 125μg/ml to 1000μg/ml. The maximum inhibition rate of D-glucosamine reachs 63.2±2.5%. Within this concentration range in vitro experiemnts find that COS achieves no significant inhibitive effects on SGC-7901 cells, however D-glucosamine produces certain inhibitive effects on HepG-2 cells with a maximum inhibition rate of 36.8±2.4%. NAG has no inhibitive effects on the growth of SGC-7901 or HepG-2 cells. In vivo studies reveal that D-glucosamine can inhibit tumor growth in S180 tumor-bearing mice and prolong survival time of H22 tumor-bearing mice, as well as protect organs of the immune system.As for experiments on anti-tumor mechanisms, it is showed that D-glucosamine could increase expression ofβ-galactosidase in SGC-7901 cells, with cell aging occurring. The morphology of SGC-7901 cells changes after treatment with D-glucosamine, with distinctive morphological changes in the nucleus and cytoplasm, included cell shrinkage, chromatin condensation, chromatin cleavage and apoptotic body formation. D-glucosamine also induces S phase cell cycle arrest in SGC-7901 cells. Calcium (Ca2+) concentration of SGC-7901 cells at different stages were observed by laser scanning confocal microscope after treatment by D-glucosamine, revealing that Ca2+ concentrations of each experimental group were significantly higher than that of the control group with the trend of 24 hours<48 hours>72 hours. Calcium concentrations in SGC-7901 cells reached their peak after treatment with D-glucosamine over 48 hours. Calcineurin expression increased after treated with D-glucosamine over 48 hours, with activity also improving significantly. However, expression of CyclinBl is found to decline. The concentrations of active oxygen and cytochrome C in SGC-7901 cells in treatment groups were found to be higher than that of-the control group, with mitochondrial membrane potential reducing. This effectively indicates that D-glucosamine is able to induce apoptosis to arrest the proliferation of tumor cells, by increasing the Ca2+ concentration in cells causing S phase arrest by activating the Ca2+ signaling pathway. Meanwhile mitochondrion damage, reduction of mitochondrial membrane potential, intra-cellular concentration of active oxygen and caspase-3 activity increased thereby inducing SGC-7901 apoptosis.As for immunological activities of COS and its derivatives, it is revealed that COS concentrations of between 100μg/ml to 50μg/ml can enhance phagocytic capacity of peritoneal macrophage of neutral red in mice (P<0.01), and remarkably promote the level of NO and TNF-a produced by macrophage. However, GlcNH2·HCl and NAG achieve no effects on macrophage phagocytosis as well as the level of NO and TNF-a within the range of 200μg/ml to 50μg/ml, compared with the control group. GlcNH2·HCl and NAG activated lymphocyte, promoted proliferation of T lymphocyte and induced T lymphocyte from G1 phase into S phase within the range of 200μg/ml to 50μg/ml. COS was found to have no increasing effects on the proliferation of lymphocyte. Ca2+ concentration in T cells were significantly higher than that of the control group after treatment with GlcNH2.HCl and NAG over 2h,4h, 6h and 24h. Intracellular Ca2+ concentration of T cells reached the highest level after treatment with GlcNH2·HCl or NAG at 6h. The expression of Calmodulin and Calcineurin in T cells increased after the treatment of NAG over 72 hours, with IL-2 secretion by T cells also increasing. These results indicate that D-glucosamine and N-acetylglucosamine have different regulative effects on cell immunity. COS affected macrophage regulation, whereas D-glucosamine and N-acetylglucosamine actived Ca2+ signal pathway of lymphocytes, which resulted in cell proliferation and IL-2 secretion.In summary, Chitooligosaccharide and D-glucosamine have significant anti-tumor and immune-regulation effects, and these double effects are beneficial for the treatment and healing of tumor patients. N-acetylglucosamine also achieved certain regulative effects on functions of lymphocyte, however, no anti-tumor effects were observed. This dissertation provides evidence for further research into the functions and use of Chitooligosaccharide, D-glucosamine and N-acetylglucosamine in tumor prevention and immune regulation.