食管癌细胞株热休克蛋白70肽复合物的提纯及抗肿瘤活性观察  

Purification of HSP70-PCs from esophageal tumor cells and detection of its anti-tumor activity to the tumor cells

作　　者：周娟[1] 李永欣[1] 孙战强[1] 李静静[1] 刘光辉[2] 祁元明[1] 

机构地区：[1]郑州大学生物工程系 [2]郑州大学第一附属医院小儿外科,郑州450052

出　　处：《郑州大学学报（医学版）》2006年第3期480-483,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：河南省科研院所专项基金资助项目0441130407

摘　　要：目的:利用抗体亲和层析法从食管癌细胞内提纯热休克蛋白70肽复合物(heatshockprotein70peptide complexes,HSP70PCs),观察其诱导的细胞毒性T淋巴细胞(CTL)对食管癌细胞的杀伤作用。方法:常规方法造成EC109细胞热休克,偶联HSP70抗体的CNBr-Sepharose4B亲和层析柱分离纯化蛋白,SDS- PAGE凝胶电泳、Westernblot检测获得的蛋白;混合淋巴细胞反应检测纯化过程中收集的蛋白粗提液、PBS洗脱蛋白及解吸附剂洗脱蛋白对食管癌细胞的杀伤活性。结果:细胞经热休克作用,裂解后获蛋白粗提液,经亲和层析柱过柱、洗脱后得到的蛋白经电泳鉴定为相对分子质量在70000左右的单一条带,且经Westernblot鉴定可与HSP70单克隆抗体结合,证实为HSP70;MTT法检测解吸附剂洗脱蛋白诱导的CTL对食管癌细胞的杀伤率为(38.10±2.78)%,明显高于蛋白粗提液(22.22±2.54)%和PBS洗脱蛋白(17.46±3.89)%,P均<0.05。结论:利用抗体亲和层析法,能够简便地从热休克处理过的食管癌细胞株中分离纯化HSP70PCs,且所获得的HSP70PCs具有较强的激活CTL杀伤食管癌细胞的生物学活性。Aim： To purify heat shock protein 70-peptide complexes （HSP70-PCs） from esophageal carcinoma cell line （EC-109） and observe the HSP70-PCs specific induced cytotoxic T lymphocyte （CTL） response on the esophageal carcinoma cells. Methods： After binding monoclonal antibody against HSP70 to CNBr-Sepharose 4B, the supernatant of 5,0 ×10^8 esophageal tumor cells （ by heat shock） lysate was applied to the column. Non-specific proteins were eluted by PBS. Then the specific protein connected with antibody was washed away by desorption buffer. The target protein was de- tected by electrophorosis and Western blot. The biological activity was detected by mixed lymphocyte reaction （MLR）. Results ： Through affinity chromatography, a single kind of protein was obtained. It was identified to be heat shock protein 70- peptide complexes which remained its higher kill rates （ （38.10 ± 2.78）% ） to EC-109 cells. The kill rate was significantly higher than other protein groups. Conclusion ： These results indicate that affinity chromatography is a simple and available method to purify HSP-PCs, which still remains stronger activity of inducing CTL to kill EC-109 cells.

关 键 词：食管肿瘤 热休克蛋白质70 分离与提纯 T淋巴细胞 细胞毒性 

分 类 号：R735.1[医药卫生—肿瘤]