三步法提取纯化肺腺癌组织中HSPgp96多肽复合物  

Three step purification of gp96 from human lung cancer line PAa

作　　者：李国[1] 王建利[1] 张玉霞[2] 单铭[2] 王俊[3] 

机构地区：[1]北京市石景山医院胸心外科,北京100005 [2]中国科学院微生物研究所 [3]北京大学人民医院胸外科

出　　处：《临床肺科杂志》2010年第7期975-977,共3页Journal of Clinical Pulmonary Medicine

摘　　要：目的建立从人肺癌组织中提取,为肺癌肿瘤免疫治疗的临床应用提供实验依据纯化热休克蛋白(HSP heatshock protein)gp96多肽复合物的方法 ;并通过体外实验对其在肺癌肿瘤免疫中的作用进行初步研究。方法运用三步蛋白纯化法从肺腺癌(PAa)组织中提取、纯化HSPgp96肽复合物,SDSPAGE、Westernblot鉴定HSPgp96多肽复合物。提取的gp96-多肽复合物与树突状细胞共孵育培养3～10天后,第10天加入到由人外周血中分离的淋巴细胞,继续培养3～7天刺激细胞毒T淋巴细胞的产生;以gp96-多肽复合物致敏淋巴细胞作为阳性对照组;以未加入任何抗原成分的DC为阴性对照。收集被致敏淋巴细胞的上清,采用IFN-γELISA检测试剂盒分析淋巴细胞所释放的IFN-γ量作为CTL反应的指标。结果应用该方法提取的蛋白质经SDSPAGE和Westernblot特异性鉴定,证实是HSPgp96多肽复合物;蛋白产率为每克PAa肺癌组织可纯化HSPgp96肽复合物约50μg。肿瘤免疫学实验发现,以gp96-多肽复合物作为肿瘤抗原负载DC,以及单独以gp96-多肽复合物作为肿瘤抗原,分别致敏淋巴细胞后均可以诱导产生CTL反应;且gp96/DC为疫苗所释放的IFN-γ量高于单独以gp96为疫苗所释放的IFN-γ量,有显著性差异(P<0.01)。结论三步蛋白纯化法不仅操作简便、重复性高,而且提取的HSPgp96肽复合物具有高获率、高纯度、高免疫效应等优点。gp96-多肽复合物能诱导出肿瘤特异性CTL反应,而将其负载DC后能激发起更强的CTL,显示出gp96-多肽复合物/DC疫苗抗肿瘤作用的广泛前景。Objective To build the method that can be used to derive and purify HSPgp96 peptide complex from human lung cancer line PAa,and investigate whether it is capable of inducing a specific cytotoxic T lymphocytes（CTL）response in vitro and provide the experimental foundation for future clinical trials of lung cancer immunotherapy.Methods Three step purification of HSPgp96 and western blot was used to derive,purify and certify HSPgp96 peptide complex from PAa tissues.DCs derived from the human peripheral blood in the presence of various cytokines were co-cultured in 3 to 7 days with the tumor-derived gp96-peptide complexes.The tester was gp96-peptide complexes/DC vaccine,and gp96-peptide complexes vaccine alone were used as positive controls,DCs without pulsing any tumor antigen were used as negative control.Then,as far as day 10th the cognate lymphocytes were added into the above DC-vaccines,after 3 to 7 days of continuing incubation,the supernate were collected and stored in-70℃.Interferon-γ （IFN-γ）secreting from activated lymphocytes was detected by using an IFN-γ ELISA Kit.Results The HSP gp96 peptide complex had a high purity and specificity.The final protein recovery was 50 μg/g tumor tissue.The results from cytotoxicity showed that DC vaccines pulsed by tumor antigens from both tester and control effectively activated autologous T lymphocytes to proliferate and secret IFN-γ.The concentration of IFN-γ induced by gp96-peptide complexes/DC were higher than that of inducing by gp96-peptide complexes vaccine,while DCs pulsed by gp96-peptide complexes could stimulate stronger immunological response than singly using gp96-peptide complexes vaccine.HSPgp96 peptide complex had the strongest immune effect in vitro.Conclusion Three step purification of HSPgp96 was simple and had a high reproducibility.HSPgp96 peptide complex obtained by this method has the merits of high purity,high specificity and strong immune effect in vitro.In vitro studies have shown that autologous tumor-derived gp96-peptide complexes

关 键 词：热休克蛋白GP96 肿瘤免疫 肺癌 

分 类 号：R734.2[医药卫生—肿瘤]