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目的以MUC1-VNTRn作为胰腺癌免疫治疗的新靶点,通过体内外实验筛选出免疫原性最强的pVAX1-MUC1-VNTRn核酸疫苗。方法构建pVAX1-MUC1-VNTRn质粒后转染未成熟树突状细胞,诱导成熟后与自体T细胞共培养,用ELISPOT检测活化分泌IFN-γ的特异性细胞毒性T细胞(CTL)数目,CytoTox检测MUC1-VNTRn特异性的CTL对Capan-2的杀伤效应,筛选出免疫原性较强的pVAX1-MUC1-VNTRn核酸疫苗。通过小鼠体内动物实验进一步证实核酸疫苗的防瘤及抗肿瘤效应。结果成功构建了pVAX1-MUC1-VNTRn质粒,并可在真核细胞表达目的蛋白。未成熟树突状细胞摄取pVAX1-MUC1-VNTRn质粒后可分化为成熟树突状细胞,经刺激后各处理组成熟的树突状细胞表达CD80、CD86、HLA-DR表面分子和分泌IL-12浓度较未经任何刺激各组的未成熟树突状细胞高(P<0.01)。负载MUC1-VNTR6和MUC1-VNTR9的树突状细胞激活自体T细胞,其分泌IFN-γ特异性T细胞数(103.0±8.5和94.3±7.7)要显著高于其他组(P<0.001)。MUC1-VNTR6和MUC1-VNTR9特异性的CTL对Capan-2杀伤效应为(40.12±3.16)%和(37.31±3.95)%,较MUC1-VNTR1强、VNTR1弱、VNTR3、VNTR4和MUC1-cDNA各组高,差异具有统计学意义(P<0.001)。小鼠体内保护性和治疗性免疫应答实验证实pVAX1-MUC1-VNTR6核酸疫苗具有最强的免疫原性,其抑制panc02-MUC1肿瘤生长能力要显著优于pVAX1-MUC1-VNTR1强、VNTR3、VNTR9核酸疫苗(P<0.01)。结论 pVAX1-MUC1-VNTRn核酸疫苗可在真核细胞表达目的蛋白。通过体内外实验筛选出pVAX1-MUC1-VNTR6核酸疫苗具有更强杀伤效应。
Objective To screen MUC1-VNTRn as the new target of immunotherapy for pancreatic cancer and screen out the most immunogenic pVAX1-MUC1-VNTRn DNA vaccine in vitro and in vivo. Methods Plasmid pVAX1-MUC1-VNTRn was constructed and transfected into immature dendritic cells. After maturation, the cells were co-cultured with autologous T cells. The number of cytotoxic T lymphocytes (CTLs) secreting IFN-γ was measured by ELISPOT. CytoTox MUC1-VNTRn-specific CTL on Capan-2 killing effect, screened strong immunogenic pVAX1-MUC1-VNTRn nucleic acid vaccine. The anti-tumor and antitumor effects of nucleic acid vaccine were further confirmed by in vivo animal experiments in mice. Results The plasmid pVAX1-MUC1-VNTRn was successfully constructed and expressed in eukaryotic cells. The immature dendritic cells can differentiate into mature dendritic cells after ingesting the plasmid pVAX1-MUC1-VNTRn. After stimulation, dendritic cells mature CD87, CD86, HLA-DR surface molecules and IL-12 secreting The concentration of immature dendritic cells was higher than that of other groups (P <0.01). Dendritic cells loaded with MUC1-VNTR6 and MUC1-VNTR9 activated autologous T cells to secrete IFN-γ-specific T cells (103.0 ± 8.5 and 94.3 ± 7.7), which were significantly higher than those of other groups (P <0.001). The killing effects of MUC1-VNTR6 and MUC1-VNTR9-specific CTLs on Capan-2 were (40.12 ± 3.16)% and (37.31 ± 3.95)%, respectively, which were stronger than those of MUC1-VNTR1, VNTR1 and VNTR3, VNTR4 and MUC1-cDNA High, the difference was statistically significant (P <0.001). In vivo protective and therapeutic immune response experiments in mice confirmed that pVAX1-MUC1-VNTR6 DNA vaccine had the strongest immunogenicity and its ability to inhibit panc02-MUC1 tumor growth was significantly better than that of pVAX1-MUC1-VNTR1, VNTR3 and VNTR9 Vaccine (P <0.01). Conclusion The pVAX1-MUC1-VNTRn nucleic acid vaccine can express the target protein in eukaryotic cells. The DNA vaccine of pVAX1-MUC1-VNTR6 was screened out by in vitro and in vivo experiments with stronger killing effect.