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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。
Objective: To study the effect of HSP70-peptide (HSP70-PC) on the activity and function of human splenic T lymphocytes (CTL). Methods: Human tongue cancer Tca8113 cells were heated at 43 ℃ for 30 min. After recovery for 12 h, the HSP70 polypeptide was purified by affinity chromatography and identified by Western blot. Purified HSP70 polypeptide was stimulated to proliferate and activate human splenic lymphocytes in vitro. Then MTT method was used to determine the cytotoxicity of tongue cancer Tca8113 cells. The morphological changes of Tca8113 cells were observed by electron microscope and phase contrast microscope. Data was t-tested using SPSS11.0 software package. RESULTS: 85 μg of purified HSP70 protein was extracted from 1g wet weight tumor cells and confirmed as HSP70 antigen peptide complex by SDS-PAGE and Western blot. HSP70-PC was extracted from tongue cancer Tca8113 cells and stimulated with human splenic lymphocytes When sensitized CTL sensitized CTLs to Tca8113 cells were 100: 1 and 50: 1, the killing rates of sensitized CTLs were (77.4 ± 2.9)% and (78.9 ± 1.9)%, respectively Significant difference (P> 0.05). The killing rate of Tca8113 induced by HSP70 was significantly different from that of non-antigen-stimulated lymphocytes (P <0.01). The morphological changes of apoptotic tongue carcinoma Tca8113 cells were observed. Conclusion: The HSP70 polypeptide derived from Tca8113 cells has a strong ability of stimulating the proliferation of T lymphocytes, stimulating the specific killing of human splenic lymphocytes and inducing the apoptosis of tumor cells.