CAJ | 학술논문

目的：将体外扩增的黏蛋白1（ MUC1） mRNA转染入成熟的树突状细胞（ DCs ），观察其体外诱导的特异性抗肿瘤效应。方法：将分离提纯的单核细胞培养诱导为DC并用流式细胞术鉴定。构建pcDNA3．1（＋）-MUC1质粒，体外转录为mRNA，电穿孔法转染DCs。定量PCR检测转染的DCs中MUC1的表达；MTT法检测T细胞增殖情况；流式细胞术检测CD8＋在T细胞的表达；LDH释放法测定细胞毒性，ELISA检测IFN-γ分泌水平。结果：流式细胞术结果表明成熟DCs标志表型的表达明显高于对照组。定量PCR结果说明转染后的DCs MUC1 mR-NA相对表达量增高。转染组DCs与T细胞按1∶10共培养时，刺激增殖能力明显高于未转染组，且CD8＋T细胞表达率高于未转染组，诱导产生特异性的细胞毒性T细胞杀伤表达MUC1蛋白的靶细胞，而未转染组的杀伤作用较弱。转染组DCs与T细胞共培养的上清中IFN-γ的分泌水平高于未转染组。结论：电穿孔法可以将MUC1 mRNA成功转染至DCs，产生特异性杀伤效应，为以MUC1为靶点的非小细胞肺癌的免疫治疗提供实验和理论依据。
목적：장체외확증적점단백1（ MUC1） mRNA전염입성숙적수돌상세포（ DCs ），관찰기체외유도적특이성항종류효응。방법：장분리제순적단핵세포배양유도위DC병용류식세포술감정。구건pcDNA3．1（＋）-MUC1질립，체외전록위mRNA，전천공법전염DCs。정량PCR검측전염적DCs중MUC1적표체；MTT법검측T세포증식정황；류식세포술검측CD8＋재T세포적표체；LDH석방법측정세포독성，ELISA검측IFN-γ분비수평。결과：류식세포술결과표명성숙DCs표지표형적표체명현고우대조조。정량PCR결과설명전염후적DCs MUC1 mR-NA상대표체량증고。전염조DCs여T세포안1∶10공배양시，자격증식능력명현고우미전염조，차CD8＋T세포표체솔고우미전염조，유도산생특이성적세포독성T세포살상표체MUC1단백적파세포，이미전염조적살상작용교약。전염조DCs여T세포공배양적상청중IFN-γ적분비수평고우미전염조。결론：전천공법가이장MUC1 mRNA성공전염지DCs，산생특이성살상효응，위이MUC1위파점적비소세포폐암적면역치료제공실험화이론의거。
AIM:To investigate the specific anti-tumor effects of mature dendritic cells ( DCs) transfected with amplified mucin 1 ( MUC1) mRNA in vitro.METHODS:DCs separated and purified from the peripheral blood mononu-clear cells were induced in vitro and then identified by flow cytometry .pcDNA3.1(+)-MUC1 plasmid was constructed and was able to transcribe MUC1 mRNA in vitro.The MUC1 mRNA was transfected into DCs by electroporation .MUC1-trans-fected DCs were used to induce T cells to be cytotoxic T-lymphocytes .Quantitative real-time PCR was performed to assess MUC1 mRNA expression in transfected DCs .The proliferation of T cells was examined by MTT assay .The proportion of CD8 +cells in the T cells was determined by flow cytometry and the specific cytotoxicity was measured by LDH assay .The secretion of IFN-γwas detected by ELISA .RESULTS: The marker gene expression in the DCs transfected with MUC 1 mRNA was significantly increased compared with control group , peaking at 24 h.The transfection group showed the higher capacity to stimulate the proliferation of T cells compared with control group when the ratio of DCs to T cells was 1∶10.The proportion of CD8 +cells in transfection group was higher than that in control group .The lethal effect of special cytotoxic T-lymphocytes on target cells in transfection group was stronger than that in control group .The level of IFN-γin the cell su-pernatant of transfection group was higher than that in control group .CONCLUSION:DCs plus MUC1 mRNA by electri-cal transfection induces specific anti-tumor effects , which provides an experiment evidence of using MUC 1 as a target for immunotherapeutic strategy against non-small cell lung cancer .