负载凋亡肿瘤细胞抗原的树突状细胞疫苗对小鼠膀胱癌的抑制作用

发布时间：2018-03-22 02:14

本文选题：树突状细胞　切入点：膀胱肿瘤　出处：《复旦大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的树突状细胞(dendritic cell, DC)是迄今为止所知道的抗原递呈功能最强的专职抗原递呈细胞(antigen presenting cell, APC),也是唯一可以激活初始免疫反应的APC,在对肿瘤产生主动免疫上起着重要的作用。树突状细胞瘤苗的制备与应用成为了肿瘤免疫治疗的研究热点。本实验研究辐射凋亡肿瘤细胞诱导的DC疫苗的制备及对C57BL/6小鼠负载膀胱肿瘤的免疫学效应。方法采用辐射法获取MB49细胞抗原并用其致敏骨髓来源的DC来制备DC疫苗。DC采用骨髓分离法从C57BL/6小鼠骨髓中分离细胞,接种于含10%胎牛血清的RPMI1640培养基中,加入重组鼠粒巨噬细胞集落刺激因子(rmGM-CSF)、重组鼠白细胞介素4(rmIL-4),体外诱导培养为未成熟DC,于第6天分别加入经辐射获取的凋亡的MB49小鼠膀胱癌细胞,刺激使其成熟,制备膀胱肿瘤疫苗。观察其形态同时流式细胞仪检测各组成熟DC表面分子CD80、CD86的表达情况。用MB49小鼠膀胱癌细胞建立荷瘤动物模型,随机分为实验组和实验对照组,于肿瘤细胞接种后第7、14天给予相应DC疫苗治疗或者PBS,每组分2亚组,分别用于测量瘤质量、体积及用于观察荷瘤小鼠存活情况。结果经鉴定建立的以DC为基础的膀胱肿瘤疫苗具有典型DC形态学和表型特征。DC疫苗扩增到第5天,通过检测表面抗体显示CDllc高表达,但是CD80,CD86以及I-A低表达,提示为未成熟DC细胞。加入.LPS共同培养后第7天通过检测CD80,CD86以及I-A提示DC细胞成熟。培养至第5天的小鼠骨髓来源的未成熟DC和经照射获得的凋亡小鼠膀胱癌细胞及无血清共培养1天后,经过流式细胞仪测定显示未成熟DC细胞成为成熟的DC细胞。经过X射线照射三天后肿瘤细胞的凋亡达到最佳数目。负载辐射凋亡肿瘤细胞抗原的DC疫苗实验组荷瘤鼠肿瘤平均体积和平均瘤质量均显著低于对照组(P0.01),生存期长于对照组,并且有2只小鼠无瘤长期存活。经DC疫苗实验组治愈的2只小鼠30天后无肿瘤生长,再次皮下接种MB49细胞观察30天仍无肿瘤发生。结论DC是功能最强大的抗原提呈细胞,负载辐射凋亡肿瘤细胞的DC疫苗对膀胱肿瘤荷瘤小鼠具有抑瘤效应和生存期延长作用。我们建立的DC疫苗诱导的特异性CTL对MB49小鼠膀胱癌细胞建立荷瘤动物模型具有的很强的细胞毒活性。负载肿瘤抗原的DC疫苗可诱导机体产生并维持长期的肿瘤记忆性细胞免疫反应,对膀胱肿瘤细胞再次攻击具有免疫保护作用,在膀胱肿瘤复发预防中具有良好应用前景。充分说明以DC为中心的肿瘤生物治疗有望提高膀胱癌综合治疗水平。
[Abstract]:Objective dendritic cells (DCs) are the most potent antigen-presenting cells known to date for antigen presenting presenting cells, and are the only ones that can activate the initial immune response. The preparation and application of dendritic cell tumor vaccine has become the focus of tumor immunotherapy. The preparation of DC vaccine induced by radiation apoptosis and the immunology of bladder tumor loaded with C57BL/6 mice. Methods MB49 cell antigen was obtained by radiometric method and DC from bone marrow was sensitized to prepare DC vaccine. DC cells were isolated from the bone marrow of C57BL/6 mice by bone marrow separation method. Inoculated in RPMI1640 medium containing 10% fetal bovine serum, The recombinant mouse granulocyte macrophage colony-stimulating factor (rmGM-CSFN) was added, and the recombinant mouse interleukin-4rmIL-4 was induced to become immature DCin in vitro. On the 6th day, the apoptotic MB49 mouse bladder cancer cells were added separately to stimulate them to mature. The bladder tumor vaccine was prepared and the expression of CD80 CD86 on the surface of mature DC in each group was detected by flow cytometry. The tumor bearing animal model was established by using MB49 mouse bladder cancer cells and was randomly divided into experimental group and experimental control group. The tumor cells were treated with corresponding DC vaccine or PBS14 days after tumor cell inoculation. Each group was divided into 2 subgroups, which were used to measure tumor quality. Results the DC based bladder tumor vaccine had typical DC morphologic and phenotypic characteristics. The DC vaccine was amplified to 5 days later, and the surface antibody was detected to show the high expression of CDllc. But CD80, CD86 and I-A are low expressed, On the 7th day after co-culture with LPS, CD80 CD86 and I-A indicated that DC cells matured. Immature DC derived from bone marrow of mice cultured to the 5th day and apoptotic mouse bladder cancer were obtained by irradiation. Cell and serum-free co-culture for 1 day, Flow cytometry analysis showed that immature DC cells became mature DC cells. After three days of X-ray irradiation, the apoptosis of tumor cells reached the best number. DC vaccine loaded with radiation apoptosis tumor cell antigen. The mean tumor volume and mean tumor mass of the tumor mice were significantly lower than that of the control group (P 0.01), and the survival time was longer than that of the control group. Two mice survived without tumor for a long time. 2 mice cured by DC vaccine had no tumor growth after 30 days, and no tumor occurred after subcutaneously inoculating MB49 cells again for 30 days. Conclusion DC is the most powerful antigen presenting cell. DC vaccine loaded with radiation apoptosis tumor cells can inhibit tumor and prolong the survival of bladder tumor bearing mice. A tumor bearing animal model of MB49 mice was established by specific CTL induced by DC vaccine. DC vaccine loaded with tumor antigen can induce the body to produce and maintain long-term tumor memory cellular immune response. It has a good application prospect in the prevention of bladder cancer recurrence, which indicates that the tumor biotherapy with DC as the center is expected to improve the comprehensive treatment level of bladder cancer.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.14

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