【作者】 王慧；

【导师】 韩宝芹；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2014， 硕士

【摘要】 壳聚糖是自然存在的唯一碱性多糖。它来源广泛，储量丰富，具有优良的生物相容性、生物可降解性和吸附性，被用做各种生物功能材料。实验所用的壳聚糖基水凝胶（CH）以改性壳聚糖为主要成分制成，是一种温敏性凝胶，37℃成胶时间最短，可在体内原位成胶。水凝胶多应用于药物载体、组织工程学等领域，但有关其抗肿瘤活性方面的报道还很少。本研究从体外、体内开展了CH对肿瘤生长的影响，并在此基础上进一步探索CH对肿瘤转移的影响，从而系统地阐述了CH的抗肿瘤效应，为CH应用于肿瘤治疗和术后恢复提供依据。体外实验采用MTT法结合显微镜观察进行CH对人正常肝细胞L02、人肝癌细胞Bel-7402、人宫颈癌细胞Hela和人胃癌细胞SGC-7901的细胞毒性评价。构建小鼠肉瘤细胞S180和小鼠肝癌细胞H22的实体瘤模型，腹腔注射方式给药，通过测定抑瘤率、免疫器官指数及相关因子水平、组织学观察来评价CH的体内抗肿瘤活性。构建H22的淋巴道转移小鼠模型，腹腔注射方式给药，从淋巴转移和肺转移情况及肿瘤转移相关因子水平方面评价CH在体内的抗肿瘤转移效应。结果显示，CH在一定浓度时，尤其是0.001g·mL-1~0.005g·mL-1时，能够抑制肿瘤细胞Bel-7402、Hela、SGC-7901的生长，最低相对增殖率为80.40%，同时促进正常细胞L02的生长，相对增殖率均大于80%，不产生细胞毒性。CH能够明显抑制体内移植性S180和H22肿瘤生长，且低剂量组效果较好，抑制率分别为39.90%和47.47%，提高免疫器官指数及细胞因子TNF-α、IL-2和IFN-γ的水平，有效杀伤肿瘤细胞，降低VEGF水平，限制血管新生。而且，采用较低剂量的CH还能够显著减轻肿瘤的肺转移和淋巴转移程度，降低促肿瘤转移相关因子VEGF和E-选择素的分泌，起到抑制肿瘤转移的效果。总之，CH能够在一定程度上抑制肿瘤细胞生长，具有良好的生物安全性；还能够增强免疫系统功能，抑制肿瘤的生长、浸润和转移。因此，CH在肿瘤切除手术中具有很好的应用前景。更多还原

【Abstract】 Chitosan is the unique alkaline polysaccharide in nature. It has extensive sourcesand abundant reserves, is widely utilized to synthesize many kinds of functionalbiomaterials for its excellent biocompatibility, biodegradability and adhesion. Thechitosan-based hydrogel (CH) used in this study, mainly made of modified chitosan, isa thermosensitive hydrogel, which takes the shortest time to gel at37℃, and can gelin situ. CH is applied in various fields such as drug-delivery systems and tissueengineering, however, its anti-tumor property is rarely reported. This research studiesthe anti-tumor activity in vitro and in vivo, then on the base of the results, develops afurther study of CH on anti-metastasis effects, so as to systematically state theanti-tumor effects of CH, providing evidences for the application of CH in tumortherapy and post-operation recovery.In vitro study, human liver cell L02, human hepatoma cell Bel-7402, humancervical carcinoma cell Hela and human gastric cancer cell SGC-7901are used toevaluate the cytotoxicity of CH by MTT assay, and the morphology of cells areobserved by inverted microscope. Transplant solid tumor models of Sarcoma180andHepatoma22are established in mice, and CH is administrated through intraperitonealinjection. Tumor inhibitory ratio, immune organ index, correlation factors levels aremeasured to investigate the anti-tumor activity of CH in vivo. Hepatoma metastasismodels of H22are established in mice, and CH is administrated throughintraperitoneal injection. Anti-metastasis effects are evaluated by pulmonary andlymphatic metastasis degrees and metastasis correlation factors levels.Results reveal that CH of certain concentration ranges, especially from0.001g·mL-1to0.005g·mL-1, could inhibite the proliferation of Bel-7402, Hela andSGC-7901cells, the lowest relative growth rate is80.40%, and promote theproliferation of L02cell, and the relative growth rates were all above80%, showingno cytotoxicity. In vivo, CH could significantly inhibite transplanted tumors of S180 and H22growth, and low-dose CH performed better, inhibition ratios are39.90%and47.47%, respectively. CH could increase immune organ index and cytokine levels ofTNF-α, IL-2and IFN-γ, kill tumor cells, lower VEGF level and restrain angiogenesis.Moreover, low-dose CH could notably decrease pulmonary and lymphatic metastasisdegrees and metastasis correlation factors levels of VEGF and E-selectin, expressesanti-metastasis effects.In conclusion, CH could inhibit tumor cells growth and promote the proliferationof normal cells to certain extend, showing good bio-safety; and enhance immunesystem function, inhibit tumor growth, infiltration and metastasis. Therefore, CHperforms good prospects in tumor removal surgery application.更多还原