gp96-肽复合物体外诱导脾淋巴细胞及其抗肿瘤效应

The anti-tumor effects of spleen T lymphocytes induced by gp96-peptide complexes in vitro

目的研究H22肿瘤细胞来源的热休克蛋白gp96-多肽复合物在体外诱导小鼠脾淋巴细胞的特异性的细胞毒性T细胞(CTL)的产生及其抗肿瘤效应。方法利用蛋白提取纯化技术、细胞培养技术、流式细胞术、CCK-8法、免疫荧光技术等。结果经鉴定获得纯化的热休克蛋白;流式细胞仪检测表明,经gp96-肽复合物诱导后的CD8T细胞比例达到近70%,远远高于对照组的35%、26%;该活化的CTL细胞在效靶比为50∶1时的肿瘤杀伤率达72%与对照组相比具有统计学意义;激光共聚焦显微镜观察证实实验诱导组的培养上清能诱导H22肿瘤细胞凋亡的形态学改变。结论肿瘤来源的热休克蛋白gp96-肽复合物能诱导小鼠脾淋巴细胞的CTL反应,该活化的CTL具有特异性抗H22肿瘤细胞的免疫保护作用并能分泌免疫活性物质诱导H22肿瘤细胞凋亡。

Objective To explore the cytotoxic T lymphocyte （CTL） induced by gp96 - peptide complexes isolated from H22 tumor cells and its anti- tumor effect in vitro. Methods Techniques for protein extraction and purification, cell culture, flow cytometry, CCK - 8 assay, laser scanning confocal microstechnic were applied. Results Purified heat shock protein gp96 were identified by SDS = PAGE gel electrophoresis and western blots analysis with FCM showed that the number of CD8^＋T cells （nearly 70%） were obviously increased after inducing with gp96- peptide complexes compared with it of control groups （35% ,26%, P 〈 0.05） ; The killing rate of CTL on target cells （ H22 tumor cells ） was 72 % at the ratio of E （Effect） to T （Target） of 50：1, which have the significant difference compared with the control groups. The morphologic change of apoptosis of H22 tumor cells which treated by culture supematant of experimental group can be observed with LSCM. Conclusioa gp96 peptide complexes can induce CTL response of spleen＇lymphocyte of mouse in vitro; Besides, those CTLs show the specific anti - tumor effect to H22 tumor cells and secrete the immunologic active material to induce apoptosis of H22 tumor cells.