体外诱导尤文肉瘤DC疫苗的特异性抗肿瘤免疫鉴定及初步临床应用报告

目的：探讨尤文肉瘤细胞总RNA转染的DC疫苗体外诱导特异性抗肿瘤免疫的能力及临床初步应用的效果。方法：分离尤文肉瘤患者外周血单核细胞体外诱导为DC细胞。Trizol法提取患者尤文肉瘤细胞总RNA,总RNA转染DC并于体外诱导特异性CTL克隆扩增。RT-PCR方法检测肿瘤总RNA加载的DC细胞内EWS-FLI1mRNA的表达。MTT法检测淋巴细胞的增殖和CTL的杀伤活性。在试验成功基础上,将成熟DC疫苗接种患者并测定免疫学OKT值。结果：RT-PCR测定显示尤文肉瘤细胞总RNA转染的DC细胞可以提呈肿瘤特异性抗原,并特异性地表达其特有序列EWS-FLI1。经总RNA转染的DC特异性表面标志及功能相关分子表达均上调,转染后的DC可显著刺激自体T淋巴细胞增殖。诱导的特异性CTL对携带EWS-FLI1抗原的靶细胞的杀伤率显著高于LAK细胞和未经转染的DC。经疫苗免疫的患者血清CD8明显升高（P〈0.05）。结论：尤文肉瘤细胞总RNA转染的DC疫苗可在体外诱导出特异性抗肿瘤免疫,并在临床初步证实可提高尤文肉瘤患者的特异性抗肿瘤免疫力。

Specific antitumor immune reaction induced by dendritic cell vaccine transfected with Ewing's sarcoma cell total RNA and preliminary report of its application in patients

Objective:To observe the ability of DC vaccine transfected with tumor cell total RNA to induce specific antitumor immunity in vitro and the primary clinical application in Ewing sarcoma patients.Methods:DCs were generated from Ewing's sarcoma patients' peripheral blood mononuclear cells(PBMC).Total RNA was isolated from Ewing's sarcoma cell by Trizol.Autologous DCs transfected with total RNA of Ewing's sarcoma cell were used to induce specific CTL proliferation.The mRNA expression of DCs tansfected with tumor total RNA was measured by RT-PCR method.T cells proliferation and specific cytotoxicity were measured by MTT method.Detected the patients OKT when were injected with DC vaccine.Results:RT-PCR showed that DCs pulsed with Ewing' sarcoma cells total RNA could present tumor specific antigen and express its characteristic sequence EWS-FLI1 specially.Transfected DCs exhibited dramatically increased expression of specific membrane markers and function-associated molecules,and were more potent in stimulating autologous T cell proliferation than that of control DCs.Specific CTLs induced by transfected DCs showed higher cytotoxicity than LAK and control DCs against EWS-FLI1 positive Ewing' sarcoma cells.The CD8 of patients who injected with DC vaccines was higher than before injection(P<0.05).Conclusion:Ewing's sarcoma patients' autologous DCs transfected with tumor total RNA are effective vaccines in stimulating specific antitumor T-cell immunity in vitro and raise the specific immunity of Ewing's sarcoma patients.