摘要
目的探讨细胞穿膜肽(cpps)介导p53致敏树突状细胞(DC)获得细胞毒性T淋巴细胞(CTL),检测其体外对结肠癌细胞株的杀伤效果。方法抽取健康志愿者外周血50ml,用淋巴细胞分离液分离外周血单个核细胞,经细胞因子诱导,获得DC细胞和T淋巴细胞。将Tat49-57-p53264-272致敏DC细胞,待DC细胞成熟后与T淋巴细胞混合诱导产生CTL,并设PBS与P53为对照。流式细胞术检测致敏前后DC细胞的表型,并采用乳酸脱氢酶(LDH)法检测抗原肽疫苗致敏DC后获得CTL对结肠癌细胞株DLD1的体外杀伤活性,同时与人白血病细胞株jurkat的杀伤作用进行比较。结果致敏前DC中CD80、CD86表达率分别为(15.9±2.1)%、(19.1±2.3)%,而致敏后分别为(17.4±1.7)%、(18.7±1.1)%,致敏前后比较差异无统计学意义,P>0.05。实验组穿膜肽可延长p53抗原多肽在DC细胞内半衰期,从而增强与DC细胞质内的MHC I类分子的结合率,并提呈到细胞表面。实验组CTL对结肠癌DLD1细胞的杀伤能力显著强于对照组,且随着效靶比的增加,杀伤活性逐渐增强(P<0.05)。Tat49-57-p53264-272多肽致敏DC活化CTL对DLD1细胞具有特异性杀伤作用,对DLD1的杀伤作用显著强于jurkat细胞(P<0.05)。结论穿膜肽可以增强p53抗原多肽的免疫原性,Tat49-57-p53264-272多肽致敏DC能有效诱导抗结肠癌DLD1细胞的特异性免疫应答。
Objective To investigate the cell penetrating peptide to carry p53 antigen pulsed dendritic cells( DC) induced antigen specific cytotoxic T lymphocytes,and in vitro the killing effect on colon cancer cell lines. Methods Mononuclear cells were extracted form 50 m L peripheral blood of healthy volunteers using lymphocyte separation liquid,using different cytokines induce cultured DC cells and T lymphocytes. Tat49-57-p53264-272 antigen peptide were prepared,and poured into DC cells. DC was mixed with T lymphocyte to induce CTL,Phenotype were respectively detected in penetratin sensitized and unsensitized DC by flow cytometry,Lactate dehydrogenase detected the activity of DLD1 colon cancer cell lines that were killed by Tat49-57-p53264-272 induced activation of CTL sensitive DC peptide vaccine,and compared with the killing effect on human leukemia cell line Jurkat.Results Sensitization of former DC CD80,CD86 expression was respectively( 15. 9 ± 2. 1) %,( 19. 1 ± 2. 3) %,while after sensitization of DC CD80,CD86 expression was( 17. 4 ± 1. 7) %,respectively( 18. 7 ± 1. 1) %,the difference was not statistically significant,P > 0. 05. The experimental group of cell penetrating peptide can prolong the half-life of P53 peptide antigen,enhanced binding and DC cell surface MHC class I molecules binding rate,and far higher than that in the control group( P < 0. 05). The experimental group cytotoxic lymphocyte on colon cancer DLD1 cell killing was significantly higher compared with the control group,and with the increase of effect or target ratio,killing activity increased gradually. The killing activity of Tat49-57-p53264-272 was significantly higher than p53264-272( P < 0. 05). Cytotoxicity of Tat49-57-p53264-272 sensitized DC polypeptide vaccine activated CTL with specific killing to DLD1 cells,the killing effect of DLD1 was significantly stronger than the killing effect on Jurkat cells( P <0. 05). Conclusion Membrane penetrating peptides can enhance the immunogenicity of P53 peptide antigen,Tat49-57-p53264-272 sensitized DC polypeptide vaccine can induce specific immune response against colon cancer DLD1 cells,and provide experimental basis for membrane penetrating peptides in further research and clinical application of tumor vaccine.
出处
《实用癌症杂志》
2015年第7期949-953,957,共6页
The Practical Journal of Cancer
