摘要
目的构建慢性髓系白血病（CML）28的树突状细胞（DC）核酸疫苗，观察其对淋巴细胞白血病的抗肿瘤效应。方法从pcDNA3．1HisA—CML28中克隆出6×His及CML28的cDNA全长，酶切后连接至真核表达载体pIRES2-增强型绿色荧光蛋白（EGFP）；从健康人外周血中分离外周血单个核细胞（PBMC），在白介素4（IL4）和粒-巨噬细胞集落刺激因子（GM—CSF）条件下诱导分化为DC；用电穿孔法将重组质粒pIRES2-EGFP—CML28导入DC，Westernblot检测His—CML28融合蛋白的表达，荧光显微镜检测EGFP的表达；用表达CML28的DC刺激同基因型淋巴细胞作为效应细胞，淋巴细胞白血病患者的PBMC作为靶细胞，以乳酸脱氢酶（LDH）释放实验测定其抗肿瘤效应。结果重组质粒pIRES2-EGFP—CML28导入DC后，能正确表达His—CML28融合蛋白及EGFP。经表达CML28的DC刺激的同基因型淋巴细胞能特异性杀伤淋巴细胞白血病患者的PBMC，杀伤率为68.3％，明显高于空白对照组（25．8％）和阴性对照组（20．7％），差异有统计学意义（P〈0．01）；而对健康人PBMC的杀伤活性较弱，杀伤率为20．5％。结论CML28能诱导特异性的细胞免疫，负载CML28的DC疫苗在体外对淋巴细胞白血病具有显著的抗肿瘤效应。
Objective To construct a vaccine encoding CML28 and investigate its anti-tumor effect in vitro. Methods The fusing gene encoding 6 x His and CML28 was amplified from pcDNA3. 1HisA- CML28 and cloned into plasmid pIRES2-EGFP. Human dendritic cells were obtained from peripheral blood mononuclear cells （PBMC） in culture with GM-CSF and IL-4. DCs were transfected with pIRES2-EGFP- CML28 by electroporation. Syngenic lymphocytes were stimulated by the transfected DCs to induce specific cytotoxic T lymphocytes （ CTLs ）. The lactate dehydrogenase （LDH） assay was used to detect the cytotoxic activity of CTL. Results DCs transfected with the recombined plasmid expressed His-CML28 and EGFP. Syngenic lymphoeytes stimulated by the transfected DCs bad specific cytotoxic activity against PBMC from patient with lymphcytic leukemia. Conclusion CML28 can elicit specific cellular immune responses, and dendritic cell-based DNA vaccine encoding CML28 can induce remarkable anti-tumor effect in vitro.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2008年第1期26-29,共4页
Chinese Journal of Oncology
基金
湖北省卫生厅资助项目（JX1B047）
湖北省科技厅攻关项目（2002AA304806）
