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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 effect of antigen-specific cytotoxic T lymphocytes (DCs) induced by DC-Tat (49-57) carrying NY-ESO-1 (155-163) antigen peptide on cell penetrating peptide (CPP) CTL) and in vitro test its killing effect on melanoma cell line A-375. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were collected and isolated from peripheral blood mononuclear cells (PBMC) by lymphocyte separation. DCs and T lymphocytes were induced by cytokines. Experimental group with Tat (49-57) -NYESO-1155-163 sensitized DC until DC mature and mixed with T lymphocytes induced CTL, and set PBS group and NY-ESO-1 (155-163) group as a control . Flow cytometry was used to detect the phenotype of DCs before and after sensitization. LDH release assay was used to detect the in vitro cytotoxicity of CTL induced by antigen peptide vaccine to melanoma cell line A-375. Human lung cancer A549 cell line, human leukemia K562 cells were compared. Results: Tat significantly increased the transmembrane capacity of NY-ESO-1 (155-163) into DC. Compared with DCs sensitized by NY-ESO-1 (155-163), CD80 / CD86 [(54.9 ± 3.3)%) of DC after sensitization of Tat 49-57 -NY-ESO- (43.8 ± 5.7)%, P <0.05] and CD40 [(42.1 ± 1.9)% vs (23.7 ± 2.8)%, P <0.05]. The majority of T cell subsets induced by Tat_ (49-57) -NY-ESO-1 (155-163) stimulated DCs were predominantly MHC class I-mediated CD3 + CD8 + cells. The cytotoxicity of CTL on Tat-49-57 -NY-ESO-1 (155-163) group was significantly higher than that of NY-ESO-1 (155-163) group. , Killing activity gradually increased (P <0.05). The cytotoxicity of CTL against NY-ESO-1 and Tat-49-57 -NY-ESO-1 (155-163) cells in A-375 cells was stronger than that in A549 and K562 cells Cells (all P <0.05). Conclusion: Tat (49-57) can enhance the immunogenicity of NY-ESO-1 (155-163) antigenic peptide, and Tat (49-57) -NY-ESO-1 (155-163) Induction of CTL specific anti-melanoma A-375 immune response.