人食管癌Eca-109细胞膜肿瘤抗原的筛选

目的:从人食管癌Eca109细胞入手,筛选出食管癌高效细胞膜肿瘤抗原的大致范围。方法:采用敏感的序列特异性引物聚合酶链反应(PCRSSP)来确定Eca109细胞人主要组织相容性复合体(HLA)基因分型,用HLA血清学分型技术筛选健康志愿者;改良的Neville法结合超滤法纯化膜蛋白并分组:<3000、3000～10000、<10000、10000～30000、30000～100000、>100000、总膜蛋白共7个组,各组抗原负载树突状细胞(DC),以DC激活的T淋巴细胞为效应细胞,Eca109细胞为靶细胞,设效靶比为81、161、321,用MTT法检测效应细胞杀伤率。结果:Eca109细胞HLA的基因分型为A03,A24;B35,B37;DRB101,DRB112;DR52;DRB3;筛选到1例表型为A03杂合子的健康志愿者。根据MTT检测,在效靶比为81、161和321时,膜蛋白相对分子质量小于10000组的肿瘤细胞杀伤率与未使用去垢剂的总膜蛋白组相比差异无统计学意义(P>0.05),而相对分子质量小于3000组的杀伤率最高,与各组相比差异均有统计学意义(P<0.05)。结论:Eca109细胞膜蛋白中存在能引起特异性的抗肿瘤细胞毒性T淋巴细胞免疫反应的肿瘤抗原,相对分子质量小于3000的膜蛋白成分中可能存在人食管癌的高效肿瘤抗原。

Screening of cytomembrane tumor antigens in human esophageal carcinoma Eca-109 cell line

Aim: To research the rough scope of high performance tumor antigens of Eca-109 cell line that can stimulate cytolytic T lymphocyte to produce anti-tumor immune response. Methods: The sequence specific primer polymerase chain reaction (PCR-SSP) method was used for HLA genotyping. The HLA alleles of Eca-109 cell line were identified. With serological HLA typing technology, the healthy adult volunteers' HLA alleles were screened to match at least one of the HLA alleles of Eca-109 cell line. The membrane proteins were purified and allocated into 7 groups. The specific cytotoxicity of CTL was obseved by MTT reduction assay, in which the ratio of effector cell (the T lymphocytes activated by DCs) to target cell (the Eca-109 cells) was set as 8:1, 16:1 and 32:1.Results: The HLA alleles of Eca-109 cell line were A*03,A*24;B*35,B*37;DRB1*01,DRB1*12;DR52;DRB3, and a healthy volunteer with heterozygote HLA-A03was found. With the ratio of effector to target of 8:1, 16:1 and 32:1, the specific cytotoxicity of CTL inMr<3 000group was significantly higher compared with other groups(P<0.05).Conclusion: In cytomembrane proteins of Eca-109cell line, there were tumor antigens that could stimulate antitumor immune response and the cytomembrane proteins lessthan 3 000 may have tumor antigens with high efficiency against human esophageal carcinoma.