HSP70多肽复合物的纯化及激活的DC疫苗抗胰腺癌体内外实验研究

发布时间：2018-04-15 02:34

本文选题：胰腺癌 + HSP70多肽复合物　；参考：《昆明医学院》2009年硕士论文

【摘要】： 目的:探讨从胰腺癌肿瘤块中分离纯化热休克蛋白70-多肽复合物(heatshock protein 70-polypeptide complexes,HSP70-PCs)的方法,研究使用HSP70多肽复合物构建树突状细胞(dendritic cell,DC)多肽疫苗,观察其在体外对CTL的增殖和活化效果。并进行免疫治疗荷瘤小鼠,观察小鼠的肿瘤大小及存活期。方法:采用低渗裂解、ConA-Sepharose亲和层析及ADP-Agarose亲和层析法,从小鼠胰腺癌(MPC83)肿瘤组织中纯化HSP70-PCs,所得蛋白经聚丙烯酰胺凝胶电泳检测,Bradford法测定蛋白浓度,计算获得率。用提纯的HSP70-PCs激活小鼠骨髓来源的树突状细胞(DC),制备树突状细胞HSP70多肽肿瘤疫苗,用MTT法检测混合淋巴细胞反应(mixed lymphocyte response,MLR)中HSP70-PCs致敏DC对CTL的增殖及活化效果;并观察DC诱导的肿瘤特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)对肿瘤细胞的特异性杀伤活性,建立MPC83荷瘤小鼠模型,并将DC接种于荷瘤小鼠皮下,观察树突状细胞HSP70多肽肿瘤疫苗对肿瘤生长的抑制作用及对荷瘤小鼠生存期的影响。结果:纯化蛋白经鉴定为分子量70KD的HSP70-PCs;平均1克湿重肿瘤组织中获得79.95μg的HSP70-PCs;HSP70-PCs在10-12μg/ml范围内可达到刺激树突状细胞最强效果,用HSP70-PCs负载的DC能增强T细胞的增殖和活化能力,其诱导的CTL对小鼠胰腺癌MPC83肿瘤细胞具有特异性杀伤作用;应用树突状细胞HSP70多肽肿瘤疫苗治疗荷瘤小鼠能显著抑制荷瘤小鼠肿瘤的生长,延长荷瘤小鼠存活期。结论:用上述层析法可分离出较高纯度、保留肿瘤抗原信息的HSP70-PCs;体外将其负载经联合细胞因子诱导培养的DC可激活T淋巴细胞,产生对该肿瘤细胞的特异性杀伤效应。体内对胰腺癌荷瘤小鼠具有显著的免疫治疗效果。
[Abstract]:Objective: to investigate the method of isolation and purification of heat shock protein 70-polypeptide complex HSP70-PCsfrom pancreatic cancer tumor, to study the construction of dendritic cell dendritic cell dendritic cell DCS vaccine using HSP70 polypeptide complex, and to observe the effect of HSP70-PCS on the proliferation and activation of CTL in vitro.The tumor size and survival period were observed.Methods: HSP70-PCswere purified from mouse pancreatic carcinoma tissue by ConA-Sepharose affinity chromatography and ADP-Agarose affinity chromatography. The protein was detected by polyacrylamide gel electrophoresis to determine the protein concentration and the yield was calculated.Dendritic cells derived from mouse bone marrow were activated with purified HSP70-PCs to prepare HSP70 polypeptide tumor vaccine from dendritic cells. The proliferation and activation of HSP70-PCs sensitized DCs in mixed lymphocyte response to MLRs were detected by MTT method.The specific cytotoxic cytotoxic T lymphocytes (CTL) induced by DC on tumor cells were observed. The MPC83 tumor-bearing mice model was established, and the DC was inoculated subcutaneously into the tumor-bearing mice.To observe the inhibitory effect of dendritic cell HSP70 polypeptide tumor vaccine on tumor growth and the survival time of tumor-bearing mice.Results: the purified protein was identified as HSP70-PCs with molecular weight of 70KD, and 79.95 渭 g HSP70-PCS70-PCs were obtained from tumor tissues with an average of 1 g wet weight, which could stimulate dendritic cells in the range of 10-12 渭 g/ml. DC loaded with HSP70-PCs could enhance the proliferation and activation of T cells.The CTL induced by CTL has a specific killing effect on MPC83 tumor cells in mice, and dendritic cell HSP70 polypeptide tumor vaccine can significantly inhibit the tumor growth and prolong the survival period of tumor-bearing mice.Conclusion: HSP70-PCs with high purity and retained tumor antigen information can be isolated by the method mentioned above, and the DC induced by the combination of cytokines can activate T lymphocytes in vitro and produce specific killing effect on the tumor cells.In vivo, there is a significant immunotherapy effect on mice bearing pancreatic cancer.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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