肿瘤热休克蛋白70-多肽复合物诱导特异性细胞毒T淋巴细胞产生的实验研究

Generation of cytotoxic T lymphocytes induced with HSP-PC70 derived from tumor cells

目的 研究肿瘤细胞的热休克蛋白 70多肽复合物 (heatshockprotein 70peptide complexes ,HSP PC70 )诱导活化肿瘤特异性细胞毒T淋巴细胞 (cytotoxicTlymphocytes,CTL)的作用及该CTL的杀瘤效应。方法 利用细胞培养 ,流式细胞技术 ,蛋白质的分离纯化技术 ,电泳技术 ,Western blot检测方法 ,T淋巴细胞的诱导及体外扩增 ,酶标技术等 ,测定肿瘤HSP70 多肽复合物诱导活化小鼠体内CTL产生的作用。结果 HcaF细胞HSP PC70的自然表达率为 6 3.5 % ,热诱导后为 75 .5 % ;HcaF细胞的HSP PC70能够诱导活化肿瘤特异性的CTL ,该CTL在效靶比例为 5 0∶1、2 5∶1、12 .5∶1时于体外对HcaF细胞的杀伤效率分别为 84.2 %± 3.2 %、47.9%± 2 .6 %和 14.8%± 3.0 % ;该CTLCD3、CD8的阳性率分别为 93%± 7% ,92 %± 8% ;将活化的CTL注入荷瘤小鼠体内可见明显的治疗作用 ,注入 10 7CTL 4次后 ,10 0 %的小鼠获长期生存。结论 肿瘤来源的HSP PC70具有强大的免疫原性 ,可活化CD8+ T淋巴细胞成为肿瘤特异性的CTL ,该CTL具有较高的体外杀瘤活性和良好的治疗作用。

Objective To investigate the killing effect of cytotoxic T lymphocytes (CTLs) induced with heat shock protein peptide complexes (HSP PC70) derived from tumor cells. Methods Cell culture, flow cytometric analysis, affinity chromatography for protein extraction and purification, SDS PAGE, Western blotting, CTL induction and expansion, and LDH release were used. Results 63.5% of the HcaF cells expressed HSP PC70 on their membrane by FCM analysis under physiological condition, and the expression was 75.5% of the stressed cells ( P <0 01). HcaF cells had high HSP70 expression and inducibility. The cytotoxic activity of the CTLs induced with HSP70 obtained by affinity chromatography from HcaF cells was 84.2%, 47.9% and 14.8% respectively at E/T ratio of 50∶1, 25∶1 and 12.5∶1. The CTLs were CD8 positive T cells. All mice which received CTL injection got long period survival (over 90 days, P <0.01), compared with control group (10.6 days). Conclusion HSP PC70 derived from tumor cells has strong immunogeneicity, and can activate CTLs with high peculiarity and killing activity. HSP PC70 induced CTL adoptive immunotherapy can be a promising treatment for human to fight cancers.