【摘要】 目的:评价修饰的CEA610D抗原肽疫苗体外抗肿瘤免疫的有效性,为其临床应用提供依据。方法:多肽固相合成法合成HLA-A2CEA修饰肽(CEA610D)、HLA-A2天然肽CEA605-613(天然肽)和HLA-A2无关肽MAGE-3(无关肽),采用同种异体混合淋巴细胞与肽共孵育的方法,诱导肽特异性细胞毒性T淋巴细胞(CTL),利用MTT法检测CTL的增殖和杀伤活性;流式细胞术观察细胞表型;RT-PCR检测细胞穿孔素的表达;ELISA法检测CTL上清中IFN-γ的水平。结果:CEA610D疫苗产生肽特异性CTL的能力最强(P<0.05),其CD8+T细胞数也高于天然肽组和无关肽组;在效靶比为40:1时,CEA610D和天然肽所诱导的CTL对CEA+HLA-A2限制性人结肠癌细胞T84的杀伤活性可达(56.7±3.73)%和(51.2±1.86)%,而3种肽特异性CTL细胞对CEA+HLA-A2人结肠癌lovo细胞杀伤活性维持在本底水平;CEA610D组的CTL所释放的杀伤相关介质穿孔素和上清中IFN-γ的水平也显著高于其余两组(P<0.01)。结论:与天然肽相比,CEA610D疫苗可打破自身表位的免疫耐受,在体外抗肿瘤免疫中更具优势。更多还原

【Abstract】 Objective:To investigated the in vivo efficacy of altered CEA610D peptide vaccine against tumor,so as to provide a basis for its clinical use.Methods:Altered CEA peptide CEA610D,natural CEA peptide CEA605-613 and MAGE-3 peptide were synthesized by polypeptide solid-phase synthesis system.The cytotoxicity T lymphocytes(CTLs) were induced by autologous mixed lymphocytes reaction implused with above peptides;proliferation and cytotoxicity of different CTLs were measured by MTT;phenotypes of the CTLs were detected by flow cytometry;expression of perforin in different CTLs were assayed by RT-PCR;IFN-γ levels in the supernatants of different CTLs were detected by ELISA.Results:CEA610D peptide more efficiently induced CTL than CEA605-613 and MAGE-3 peptide did(P<0.05).The number of CD8+T cells in CEA610D group was significantly larger than those in CEA605-613 and MAGE-3 groups(P<0.05).When the E∶T was 40∶1,the cytotoxicity of CTLs induced by CEA610D and CEA605-613 against CEA+HLA-A2+ T84 cells were(56.7±3.73)% and(51.2±1.86)%,respectively.But the CTL cytotoxicities induced by the three peptides against CEA+HLA-A2-Lovo cells were all at the background level.Perforin expression and IFN-γ level of CTLs in CEA610D group were significantly higher than those in the other two groups(P<0.01).Conclusion:Compared with natural CEA605-613 peptide,altered CEA610D peptide can effectively break the immune tolerance to self peptide,and thus has a stronger anti-tumor activity in vitro.更多还原