CAJ | 학술논문

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。
목적：이MUC1-VNTRn작위이선암면역치료적신파점，통과체내외실험사선출면역원성최강적pVAX1-MUC1-VNTRn핵산역묘。방법구건pVAX1-MUC1-VNTRn질립후전염미성숙수돌상세포，유도성숙후여자체T세포공배양，용ELISPOT검측활화분비IFN-γ적특이성세포독성T세포（CTL）수목， CytoTox?검측 MUC1-VNTRn 특이성적 CTL 대 Capan-2적살상효응，사선출면역원성교강적pVAX1-MUC1-VNTRn핵산역묘。통과소서체내동물실험진일보증실핵산역묘적방류급항종류효응。결과성공구건료 pVAX1-MUC1-VNTRn 질립，병가재진핵세포표체목적단백。미성숙수돌상세포섭취pVAX1-MUC1-VNTRn질립후가분화위성숙수돌상세포，경자격후각처리조성숙적수돌상세포표체CD80、CD86、HLA-DR표면분자화분비IL-12농도교미경임하자격각조적미성숙수돌상세포고（P＜0.01）。부재MUC1-VNTR6화 MUC1-VNTR9적수돌상세포격활자체T세포，기분비IFN-γ특이성T세포수（103.0±8.5화94.3±7.7）요현저고우기타조（P＜0.001）。MUC1-VNTR6화MUC1-VNTR9특이성적CTL대Capan-2살상효응위（40.12±3.16）%화（37.31±3.95）%，교MUC1-VNTR1강、VNTR1약、VNTR3、VNTR4화MUC1-cDNA각조고，차이구유통계학의의（ P＜0.001）。소서체내보호성화치료성면역응답실험증실pVAX1-MUC1-VNTR6핵산역묘구유최강적면역원성，기억제 panc02-MUC1종류생장능력요현저우우pVAX1-MUC1-VNTR1강、VNTR3、VNTR9핵산역묘（P＜0.01）。결론 pVAX1-MUC1-VNTRn 핵산역묘가재진핵세포표체목적단백。통과체내외실험사선출pVAX1-MUC1-VNTR6핵산역묘구유경강살상효응。
Objective MUC1-VNTRn served as a new target for pancreatic cancer immunotherapy in the present study. We intended to screen out the most powerful pVAX1-MUC1-VNTRn DNA vaccine which showed strongest immunogenicity in vitro and vivo. Methods pVAX1-MUC1-VNTRn were constructed and transfected into MUC1 negative cells. The target protein of MUC1-VNTRn could be detected by Western Blot. Dendritic cell preparation and research of biological characteristics. pVAX1-MUC1-VNTRn were transfected into dendritic cell. The stimulated mature dendritic cells were co-cultured with autologous T cells, and then we used the ELISPOT to detect the IFN-γsecreting CTL. We detected the cytotoxic effect of the antigen-specified CTL to Capan-2 cells in vitro. Finally, we used the the protective and therapeutic immune response in mice to confirm the immunogenicity of DNA vaccine. Results pVAX1-MUC1-VNTRn were successfully constructed, and the target protein can be expressed in eukaryotic cells. The immature dendritic cells can intake pVAX1-MUC1-VNTRn plasmid, and then differentiate into mature dendritic cells. Expression of CD80, CD86, HLA-DR and secretion of IL-12 by stimulated mature dendritic cells were higher than that of unstimulated immature dendritic cells (P<0.01). The ELISPOT result showed that the number of IFN-γ secreting CTL in MUC1-VNTR6 (103.0±8.5) and VNTR9 (94.3±7.7) group were more than the other group (P<0.001). Cytotoxic effect of the MUC1-VNTR6 and VNTR9 specified CTL to Capan-2 cells were (40.12±3.16)%and (37.31±3.95)%, which was higher than that of the other group (P<0.001). Protective and therapeutic immune response in mice showed that pVAX1-MUC1-VNTR6 had the strongest immunogenicity. Conclusions pVAX1-MUC1-VNTRn can express target protein in eukaryotic cells. We screen out the pVAX1-MUC1-VNTR6 DNA vaccine which shows the strongest immunogenicity in vitro and vivo.