HSP70多肽复合物介导的细胞毒性T淋巴细胞对Tca8113细胞的免疫杀伤作用

The effect of HSP70 peptide complexes during hyperthermia and immunotherapy mediated by cytotoxic T lymphocytes on Tca8113 cells

目的:研究HSP70多肽(HSP70peptide complexes,HSP70-PC)对人脾细胞毒T淋巴细胞(CTL)活性及功能的影响。方法:人舌癌Tca8113细胞在43℃条件下加热30min,恢复12h后,用亲和层析方法提纯HSP70多肽,并用Western印迹鉴定;将提纯的HSP70多肽体外刺激人脾淋巴细胞扩增、活化,然后用MTT法测定其对舌癌Tca8113细胞的杀伤作用,并用电镜和相差显微镜观察其培养上清舌癌Tca8113细胞的形态学变化。采用SPSS11.0软件包对数据进行t检验。结果:1g湿重的肿瘤细胞可提取纯化HSP70蛋白85μg,通过SDS-PAGE和Western印迹证实为HSP70抗原肽复合物;提取舌癌Tca8113细胞的HSP70-PC与体外免疫扩增的人脾淋巴细胞形成致敏CTL,当致敏CTL与Tca8113细胞效靶比为100:1和50:1时,致敏CTL的杀伤率分别为(77.4±2.9)%和(78.9±1.9)%,2组间无显著差异(P>0.05)。HSP70诱导的CTL对Tca8113的杀伤率与非抗原刺激的淋巴细胞组比较,有显著性差异(P<0.01),受攻击的舌癌Tca8113细胞出现凋亡的形态学变化。结论:Tca8113细胞来源的HSP70多肽具有较强的刺激T淋巴细胞增殖的能力,能刺激人脾淋巴细胞产生特异性杀伤肿瘤细胞的效应,并能介导肿瘤细胞凋亡。

PURPOSE： To investigate the killing effect of cytotoxic T lymphocytes（CTLs） by HSP70-PC antigen peptides purified from TcaS113 cells. METHODS： HSP70 associated TcaS113 cells tumor peptides were purified from tumor cell pellet treated in water bath （43℃/30min） by ConA-Sepharose column,ADP-Agarose column,DEAE ino-exchange chromatography. The purified fractions tested by SDS-PAGE,immunoblotted with anti-HSP70 mAb.Its immunoprotective effect of antitumor was assessed by active immunoprotective assays; antigen peptides mixtures were prepared from human squamous tongue cancer Tca8113 cell lines and then bound with HSP70 in vitro. Activation and proliferation of CTLs were observed after stimulation with HSP70-peptide complexes. The cytotoxicity of the activated CTLs to different target cells was assayed. The data were analyzed with SPSS11.0 software package for Student＇s t test. RESULTS： HSP70 tumor peptide was purified from heated Tca8113 cells, which was a high purity protein with 70 kD molecular weight and integrated with anti-HSP70 mAb; CTLs immunized with the protein protected Tca8113 cells from challenging in vitro. Tumor-specific CTLs activated on the condition of effector：target （=100：1,50：1） reached about 77%-80% ,there was no significant difference between group（100：1） and group（50：1）（P〉0.05）;however,there was significant difference between both the two groups and the other group （P〈0.01）. CONCLUSIONS： The highly purified HSP70-PC can be obtained successfully by using the affinity chromatography and DEAE ion-exchange chromatography. The HSP70-PC from tumors can make the lymphocyte become into CTL more effectively. Apoptosis of Tca8113 cells is induced by CTL via soluble factors,these CTLs show prominent killing effect on the target tumor cells.