【作者】 荆雪宁；

【导师】 武继彪；

【作者基本信息】 山东中医药大学 ， 中西医结合基础， 2013， 博士

【摘要】 目的：本研究主要分两部分。第一部分是黄芪多糖诱导的DC肿瘤疫苗体外抗肿瘤作用的实验研究，主要观察黄芪多糖诱导DC成熟及对DC免疫功能的影响；第二部分是黄芪多糖诱导的DC肿瘤疫苗对荷瘤小鼠免疫治疗效应的研究，主要观察黄芪多糖诱导的DC肿瘤疫苗对荷瘤小鼠的抗肿瘤效果，并探讨黄芪多糖诱导的DC肿瘤疫苗体内抗瘤的机制，为进一步开拓中药治疗肿瘤的途径提供实验和理论依据。方法：1.人外周血单个核细胞用IL-4、GM-CSF诱导分化为未成熟DC，加入黄芪多糖促进成熟，并设细胞因子组、黄芪多糖+细胞因子组，显微镜下连续观察形态变化，用FACS检测各组DC表型。2.成熟DC以肿瘤抗原致敏，与同种异体T细胞共培养，用MTT法检测T淋巴细胞增殖功能，用ELISA法检测共培养上清IL-12、IFN-γ水平。3.经DC激活的CTL细胞与肿瘤细胞共培养，用LDH释放法检测CTL对靶细胞特异性杀伤能力。4.小鼠骨髓来源的DC以黄芪多糖诱导成熟，负载S180肿瘤抗原，制备肿瘤疫苗。建立S180荷瘤小鼠模型，随机分成模型对照组、阳性对照组、黄芪多糖组和细胞因子组。给S180荷瘤小鼠进行免疫治疗，观察抑瘤率和小鼠生存时间。5.小鼠经眼眶采血，分离血清。ELISA法检测小鼠血清IL-12、TNF-α的水平。6.取小鼠脾脏，分离淋巴细胞，用MTT法检测小鼠脾脏淋巴细胞转化率，FACS检测小鼠Th1/Th2亚群。结果：1.黄芪多糖诱导的DC经形态学观察和表型鉴定，符合成熟DC的特征，黄芪多糖可以诱导DC成熟。2.黄芪多糖诱导的DC激活的CTL体外特异性杀伤肿瘤细胞的能力与TNF-α诱导的DC无显著性差异，提示黄芪多糖诱导的DC可以有效的激活CTL细胞，发挥特异性抗肿瘤作用。3.黄芪多糖诱导的DC能刺激同种异体T淋巴细胞增殖，并提高DC与T细胞共培养上清IL-12、IFN-γ的水平，提示黄芪多糖诱导的DC可以有效的给T细胞提呈抗原，提供活化信号，促进T细胞增殖，并分泌IFN-γ；同时，有效的抗原提呈也促使DC活化，分泌抗肿瘤细胞因子IL-12。4.黄芪多糖诱导的DC疫苗可减缓肿瘤生长速度，显著发挥体内抑瘤作用，延长荷瘤小鼠的生存时间。5.黄芪多糖诱导的DC疫苗能促进S180荷瘤小鼠分泌TNF-α、IL-12，通过抗肿瘤细胞因子途径发挥抗肿瘤作用。6.黄芪多糖诱导的DC疫苗可以显著提高小鼠脾脏淋巴细胞转化率，促使Th1/Th2向Th1转化，提示黄芪多糖诱导的DC疫苗可以增强机体免疫功能，通过调节Th1/Th2失衡状态向Th1方向转化，增强机体的细胞免疫功能。结论：黄芪多糖可以体外诱导DC成熟。经黄芪多糖诱导成熟的DC疫苗可有效的向T细胞提呈抗原，促进T细胞的增殖和活化，增加抗肿瘤细胞因子的分泌，激活CTL特异性抗肿瘤的能力，显示了良好的体外抗肿瘤效果。黄芪多糖诱导的DC疫苗可以通过增加荷瘤小鼠T细胞增殖能力、促进细胞因子TNF-α、IL-12的产生、诱导Th1/Th2向Th1转化等机制增强机体抗肿瘤免疫功能，减缓荷瘤小鼠瘤体生长速度，发挥延长生命作用。更多还原

【Abstract】 Objection: The present work comprises two parts. In the first part,theantitumoractivity induced by dendritic cells is studied in vitro to observe the influence ofastragalus polysaccharides on maturation and immunologic function of dendritic cells. Inthe second part, the antitumoractivity induced by dendritic cells is studied in tumor-bearingmice, to investigate the anti-tumor effect of dendritic cells and explore the mechanism.Methods:1. The dendritic cells from human peripheral blood are cultured and induced with IL-4,GM-CSF into immature dendritic cells, which are randomly divided in to3groups. GroupA is treated with astragalus polysaccharides, Group B with TNF-αand Group C withastragalus polysaccharides and TNF-α. The morphotype of dendritic cells is identified byinverted optical microscope and the phenotype of cultured dendritic cells (CD80, CD86) isidentified by flow cytometry.2. Mature dendritic cells loaded with tumor antigens are co-cultured with alloreactiveT cells. MTT is used to detect the effect of astragalus polysaccharides on the function ofdendritic cells in stimulating the proliferation of T cells. ELISA is used to detect the levelof IL-12, IFN-γ in cells culture supernatant.3. Dendritic cells are loaded with tumor antigen and co-cultured with alloreactive Tcells to induce generation of tumor specific cytotoxic T cells(CTL). Then CTL areco-cultured with SGC-7901tumor cells. The killing activity of CTL to SGC-7901cells wastested by LDH release assay.4. Dendritic cells derived from mouse bone marrow are induced maturation andloaded with S180tumor antigen to prepare tumor vaccine. Tumor-bearing mice are divided in to4groups, Group A is injected with NS, Group B with CTX, Group C with dendriticcells induced by astragalus polysaccharides and Group D with dendritic cells induced byTNF-α. The tumor size and survival of the mice are observed.5. ELISA assay is usded to detect the number of IL-12, TNF-α in serum of tumorbearing mice.6. The proliferation of splenocytes is determined by using MTT assay and Th1/Th2was detected by flow cytometry.Results:1. According to the morphology and phenotype, dendritic cells induced by Astragaluspolysaccharides are in accordance with the characteristics of mature dendritic cells.Astragalus polysaccharide can induce dendritic cells maturation.2. CTL activated by dendritic cells can specifically kill tumor cells and the killactivity has no significant difference between the Astragalus polysaccharides group andTNF-α group, suggesting that dendritic cells induced by Astragalus polysaccharides caneffectively activate CTL cells to exert the function of specific anti-tumor.3. Dendritic cells induced by Astragalus polysaccharides can reinforce proliferation ofalloreactive T cells and increase the number of IL-12, IFN-γ in supernatant, suggesting thatDC induced by Astragalus polysaccharides can effectively present antigen to T cell,provide the activation signal, promote the proliferation of T cells and secretion of IFN-γ; atthe same time, dendritic cells are activated to secret IL-12.4. DC vaccine induced by Astragalus polysaccharide can significantly exert antitumoreffect in vivo and prolong the survival time of mice bearing tumor.5. DC vaccine induced by Astragalus polysaccharides can promote the secretion ofTNF-α and IL-12in S180tumor-bearing mice, suggesting that DC vaccine exertsanti-tumor effect through anti-tumor cytokine pathway.6. DC vaccine induced by Astragalus polysaccharide can significantly improve themouse spleen lymphocyte transformation rate, adjust Th1/Th2to drift to Th1, suggestingthat DC vaccine induced by Astragalus polysaccharide can enhance immune function andcell immunity by making the Th1/Th2imbalance transformation to Th1direction.Conclusion: DC vaccine induced by Astragalus polysaccharides can effectivlypresent tumor antigen to T cell, and stimulate T cell to proliferate and activate, increase thesecretion of cytokines, activate CTL to exert specific anti-tumor ability, showing goodantitumor effect in vitro. DC vaccine induced by Astragalus polysaccharides can enhance the antitumor immunity of tumor-bearing mice by promoting T cell proliferation,increasing the secretion of TNF-α, IL-12, inducing Th1/Th2balance to Th1conversion.Finally, DC vaccine can slow down tumor growth speed and prolong the life oftumor-bearing mice.更多还原