MUC1/Y基因真核表达载体构建及MUC1、MUC1/Y体外修饰DC诱导特异性抗肿瘤免疫应答  

The construction of eukaryotic vectors that express MUC1/Y and the outcome of modifying dendritic cells to induce specific immunoresponse

作　　者：刘虹[1] 任秀宝[1] 魏枫[1] 尹志伟[1] 郝希山[1] 

机构地区：[1]天津医科大学附属肿瘤医院免疫室,300060

出　　处：《中华微生物学和免疫学杂志》2004年第7期533-537,共5页Chinese Journal of Microbiology and Immunology

摘　　要：目的　构建MUC1 Y全长cDNA真核表达载体 ,并以MUC1及MUC1 Y真核表达载体修饰树突状细胞 (DC)诱导特异性抗肿瘤免疫。方法　构建MUC1 YDNA真核表达载体 ,选取 10例HLA A2乳腺癌患者 ,体外IL 4、GM CSF联合诱导DC ,并以pCDNA3.1 MUC1和pCDNA3.1 MUC1 Y转染DC ,同时以空载体为对照 ,以pIRES2 EGFP MUC1 Y观察转染效率 ,转染后DC与自体T细胞混合培养 ,诱导CTL。以MCF 7为特异性靶细胞 ,Raji为非特异性靶细胞 ,通过乳酸脱氢酶释放实验检测杀伤活性 ,以annexinⅤ FITC检测特异性CTL诱导靶细胞凋亡的情况。以ELISA法测定基因修饰后DC刺激自体T细胞分泌IFN γ的能力。结果　酶切鉴定及测序结果表明成功构建了MUC1 Y真核表达载体pIRES2 EGFP MUC1 Y和pCDNA3.1 MUC1 Y。pIRES2 EGFP MUC1 Y转染效率基本为 10 %左右 ,DC中MUC1表达率为 8%。杀伤实验表明T DC pCDNA3.1 MUC的杀伤活性为 6 4 % ,T DC pCDNA3.1 MUC1 Y为 4 6 %。这两组间差异有显著性 ,同时与对照组比较差异均有显著性。annexinⅤ FITC标记实验显示 ,T DC pCDNA3.1 MUC1对靶细胞的杀伤和诱导凋亡的能力显著高于T DC pCDNA3.1 MUC1 Y及对照组。基因修饰DC刺激自体T细胞分泌IFN γ的能力显著升高。结论　构建的真核表达载体pIRES2 EGFP MUC1Objective To develop MUC1 and MUC1/Y gene therapy for breast cancer,we constructed the eukaryotic expression vectors that express a full length MUC1/Y cDNA. To modify DC with these vectors in order to induce specific immunoresponse. Methods To collect the MUC1/Y full-length cDNA from breast cancer cell line T-47D and then clone it into pIRES2-EGFP expressing green fluorescence protein and eukaryotic expression vector pCDNA3.1 respectively. To selected 10 breast cancer patients with HLA-A2 phenotype,and then to induce DC by rhIL-4 and GM-CSF in vitro. MUC1/Y-pCDNA3.1 or MUC1-pCDNA3.1 were used to pulse DC,then co-cultured DC with T cell to induce CTL. At the same time,transfected DC with MUC1/Y-pIRES-EGFP to show the transfection efficiency. MCF-7,the breast cancer cell line,which expresses MUC1/Y,MUC1,and HLA-A2,was used as target cell;the cytolytic of specific CTL was measured by LDH-releasing assay. IFN-γ was quantified by ELISA. Results The results of the sequencing demonstrated that we have cloned MUC1/Y successfully. The transfection efficiency of plasmid was about 10%. The cytolytic activity of T-DC-MUC1-pCDNA3.1 was about 64%,and the cytolytic activity of T-DC-MUC1/Y-pCDNA3.1 was about 32%. There was a significant difference between these results and those of control group respectively. The results of annexin Ⅴ-FITC experiments showed that T-DC-MUC1-pCDNA3.1 induced apoptosis of specific target cell. Conclusion Constructed eukaryotic vector pIRES2-EGFP-MUC1/Y that contains full length MUC1/Y cDNA can be used to study the transfection efficiency and select the positive clone;MUC1-pCDNA3.1 and MUC1/Y-pCDNA3.1 can induce powerful CTL immunoresponse,especially MUC1-pCDNA3.1.

关 键 词：MUC1基因 Y基因 真核表达载体 体外修饰 DC诱导 特异性 抗肿瘤免疫应答 基因疫苗 乳腺癌 

分 类 号：R730.3[医药卫生—肿瘤]