不同类型肿瘤抗原致敏的树突状细胞体外诱导前列腺癌特异性CTL的研究

Induction of prostate cancer-specific CTLs with dendritic cells pulsed by different types of tumor antigens

目的:研究肿瘤冻融抗原及弱酸洗脱提取的肿瘤抗原肽分别冲击致敏树突状细胞(dendritic cells,DCs)体外诱导前列腺癌特异性细胞毒性T淋巴细胞(CTL)的效应。方法:采用反复冻融法和弱酸洗脱法,分别获取前列腺癌细胞株PC-3的肿瘤抗原,应用重组人GM-CSF、IL-4体外培养诱导外周血单个核细胞获得DC,并分别负载两种前列腺癌肿瘤抗原,检测其体外激活肿瘤特异性CTL的能力,用LDH释放法检测其对PC-3的体外杀伤效应。结果:用枸橼酸-磷酸盐缓冲液洗脱或反复冻融PC-3(2×107)后获得的肿瘤抗原蛋白含量分别为(312.2±7.9)μg和(963.0±25.3)μg,负载两种前列腺癌细胞抗原的DCs致敏的CTL对PC-3细胞有明显的杀伤作用,而且负载弱酸洗脱提取的肿瘤抗原肽的DCs致敏的特异性CTL具有更明显的杀伤作用[(60.4±5.52)%vs(43.7±4.11)%,P<0.01)]。结论:弱酸洗脱和反复冻融方法都可以有效获取前列腺癌抗原,用于体外致敏DCs后,均具有活化CTL的能力,而且弱酸洗脱提取的肿瘤抗原肽更为有效。

Objective：To study the effectiveness of freeze-thaw antigens and acid eluted peptide antigens extracted from tumor cell-pulsed dendritic cells （DC） in inducing prostate cancer-specific cytotoxic T lymphocytes （CTL） in vitro.Methods：Tumor antigens were extracted from the prostate cancer cell line PC-3 with the repeated freeze-thaw and weak acid elution methods.Peripheral blood mononuclear cells were cultured with recombinant human GM-CSF and IL-4 for inducing DCs in vitro.Then the DCs were pulsed with the two kinds of prostate cancer tumor antigens respectively and cultured with T cells for inducing CTLs.The activity of the tumor-specific CTLs were detected by LDH release assay.Results：The protein content in the tumor antigens obtained from PC-3 （2 × 107） by citric acid-phosphate buffer elution and that by the repeated freeze-thaw method were （212.2 ± 7.9） μg and （963.0 ± 25.3） μg,respectively.The two kinds of prostate cancer antigens-pulsed DCs had a significant role in inducing the PC-3 cell-specific CTLs,and the CTLs induced by acid-eluted peptide antigen-pulsed DCs exhibited an even more significant tumor-specific cytotoxicity than those induced by repeated freeze-thaw （[60.4 ± 5.52]% vs [43.7 ± 4.11]%,P0.01）.Conclusion：Both the weak acid elution and repeated freeze-thaw methods for extracting prostate cancer antigens can be used for in vitro sensitization of DCs.The DCs pulsed by either of the two kinds of antigens can activate CTLs,and the antigens extracted by weak acid elution are even more effective.