穿膜肽介导的NY-ESO-1_(155-163)诱导CTL对黑素瘤A-375细胞的杀伤活性  

Cytotoxic effect of CTLs toward melanoma A-375 cells induced by cell penetrating peptide-fused NY-ESO-1_(155-163)

作　　者：李文丽[1] 贾原[1] 赵晴[1] 李芳[1] 靳保利 张俊萍[1] 

机构地区：[1]山西医学科学院山西大医院肿瘤生物治疗科,山西太原030032

出　　处：《中国肿瘤生物治疗杂志》2015年第6期710-715,共6页Chinese Journal of Cancer Biotherapy

基　　金：山西省生物治疗示范平台项目资助(No.2014091105-0101)~~

摘　　要：目的:探讨穿膜肽(cell penetrating peptide,CPP)Tat_(49-57)携带NY-ESO-1_(155-163)抗原肽致敏DC后诱导获得抗原特异性细胞毒性T淋巴细胞(cytotoxic lymphocyte,CTL),并体外检测其对黑素瘤细胞株A-375的杀伤效果。方法:采集健康志愿者外周血50 ml,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和T淋巴细胞。实验组用Tat_(49-57)-NYESO-1155-163致敏DC,待DC成熟后与T淋巴细胞混合诱导产生CTL,并设PBS组和NY-ESO-1_(155-163)组作为对照。流式细胞术检测致敏前后DC的表型,乳酸脱氢酶(LDH)释放法检测抗原肽疫苗致敏DC后诱导的CTL对黑素瘤细胞株A-375的体外杀伤活性,同时与对人肺癌A549细胞株、人白血病K562细胞的杀伤作用进行比较。结果:Tat显著提升NY-ESO-1_(155-163)进入DC的穿膜能力。与NY-ESO-1_(155-163)致敏的DC相比,Tat_(49-57)-NY-ESO-1_(155-163)致敏后DC的CD80/CD86[(54.9±3.3)%vs(43.8±5.7)%,P<0.05]和CD40[(42.1±1.9)%vs(23.7±2.8)%,P<0.05]的表达率显著升高。Tat_(49-57)-NY-ESO-1_(155-163)刺激DC后诱导培养的T细胞亚群主要以MHC-Ⅰ类分子介导的CD3+CD8+细胞为主。Tat_(49-57)-NY-ESO-1_(155-163)组CTL对A-375细胞的杀伤能力显著高于NY-ESO-1_(155-163)组,且随着效靶比的增加,杀伤活性逐渐增强(P<0.05)。A-375细胞高表达NY-ESO-1,Tat_(49-57)-NY-ESO-1_(155-163)组CTL对A-375细胞具有特异性杀伤作用,杀伤作用显著强于A549和K562细胞(均P<0.05)。结论:Tat_(49-57)可以增强NY-ESO-1_(155-163)抗原多肽的免疫原性,Tat_(49-57)-NY-ESO-1_(155-163)多肽致敏DC能有效诱导CTL抗黑素瘤细胞A-375的特异性免疫应答。Objective: To investigate the cytotoxic effect of cytotoxic lymphocytes（ CTLs） activated by Tat49-57-NYESO-1155-163 sensitized dendritic cells on melanoma A-375 cells. Methods: We collected the peripheral blood（ about 50ml） from healthy volunteers and isolated mononuclear cells by using lymphocyte separation medium. The cells were treated with cytokines to produce dendritic cells and T lymphocytes. After sensitized with the Tat49-57-NY-ESO-1155-163 peptide,the dendritic cells were co-cultured with the T lymphocyte cells to generate antigen-specific CTLs. Phenotypes of the dendritic cells were examined by flow cytometry. The antigen-specific cytotoxic activity of the CTLs against A-375 melanoma cells in vitro was assessed by the lactate dehydrogenase（ LDH） method. Human lung cancer A549 cells and leukemia K562 cells were used as controls. Results: The expression rate of CD80 / CD86 in dendritic cells sensitized with Tat49-57-NY-ESO-1155-163was（ 54. 9 ± 3. 3） % significantly higher than these sensitized with NY-ESO-1155-163（ [43. 8 ± 5. 7]%,P < 0. 05）.There were also significant increase of CD40 expression（ [42. 1 ± 1. 9]% vs [23. 7 ± 2. 8]%,P < 0. 05） in Tat49-57-NYESO-1155-163 sensitized dendritic cells. These results indicated that the Tat fragment（ 49- 57） significantly improved the cell penetrating ability of NY-ESO-1155-163. The T lymphocytes activated by the Tat49-57-NY-ESO-1155-163 sensitized DC were mainly CD3+CD8+cells. The cytotoxic activity of the CTLs induced by Tat49-57-NY-ESO-1155-163-sensitized dendritic cells toward A-375 melanoma cells was significantly higher than these induced with NY-ESO-1155-163-sensitized dendritic cells（ P< 0. 05）. The CTLs were also specific as they killed NY-ESO-1-expressing A375 more efficient than A549 cells and K562cells（ P < 0. 05）. Conclusion: Tat49-57 cell penetrating peptide can enhance the immunogenicity of the peptide NY-ESO-1155-163. Tat49-57-NY-ESO-1155-163polypeptide-sensitized dendritic cells can effectively induce the s

关 键 词：黑素瘤 树突状细胞 细胞穿膜肽 肿瘤免疫治疗 

分 类 号：R739.5[医药卫生—肿瘤]