MUC1/Y基因体外冲击树突状细胞诱导抗瘤免疫反应

The clinical senses of MUC1/Y gene expressing in gastrointestinal cancer and MUC1/Y modified DC inducing anti-tumor response

目的 :制备含MUC1 YcDNA质粒转染的树突状细胞 (DC) ,体外诱导杀伤细胞 ,研究其治疗消化道肿瘤的效果。方法 :构建MUC1 YcDNA真核表达载体pIRES2 EGFP MUC1 Y、pcDNA3 1 MUC1 Y。以pcDNA3 1 MUC Y电转染 8例HLA A2 ( + )消化道肿瘤患者单个核细胞衍生的DC后 ,与自体T细胞混合培养 ,诱导CTL(T pcDAN3 1 MUC1 Y)。以SW6 2 0细胞 [HLA A2 ( + ) 、MUC1 Y( + ) ]为特异性靶细胞 ,Raji细胞 [HLA A2 ( - ) 、MUC1 Y( - ) ]和Lovo细胞 [HLA A2 ( - ) 、MUC1 Y( + ) ]为非特异性靶细胞 ,通过乳酸脱氢酶 (LDH)释放实验测定杀伤活性 ,ELISA法检测基因修饰后DC刺激自体T细胞产生IFN γ的能力 ,并以AN NEXINV FITC试剂盒检测特异性CTL诱导靶细胞凋亡情况。结果 :pIRES2 EGFP MUC1 Y转染效率为 8%左右。T pcDAN3 1 MUC1 Y诱导的杀伤作用显著高于T pcDNA3 1[pcDNA3 1(+)修饰DC诱导的CTL]和T IL 2 (IL 2刺激外周血单个核细胞产生的CTL) ,P <0 0 5。而且T pcDNA3 1 MUC1 Y对靶细胞的杀伤和诱导凋亡的能力显著高于对照组。基因修饰后的DC能刺激自体T细胞分泌高水平IFN γ ,与未转染的DC相比具有显著差异 (P <0 0 5 )。结论 :成功构建MUC1 Y全长cDNA真核表达载体。pIRES2 EGFP MUC1 Y可用于真核细胞转染 ,通过观察转?

Objective:For developing the study of MUC1/Y vaccine,constructed th eukaryotic expression vectors expressing MUC1/Y.Used it to modify the DC inducint CTL in order to treat the gastrointestinal cancers.Methods:Obtained the MCU1/Y cDNA full length cloned it into pIRES2 EGFP that expressing green fluorescence protein and pcDAN3 1+ respectively.Selected 8 cases of gastrointestinal cancer whose HLA phenotype was A2,inducing DC using rhIL 4 combined with GM CSF in vitro.Transfected DC with pcDAN3.1 MUC1/Y then co cultured DC with T cell to induce CTL (named as T pcDAN3 1 MUC1/Y).At the same time,used pIRES2 EGFP MUC1/Y to detect transfection efficiency.SW620 (HLA A2+,MUC1/Y+),the gastric cancer cell line was used as specific target cell;Lovo(HLA A2 ,MUC1/Y+)and Raji(HLA A2 ,MUC1/Y )were used as unspecific target cells.The cytolytic of specific CTL was measured by LDH releasing assay.The apoptosis of target cells were detected by ANNEXIN V FITC kit.The ability of IFN γ releasing of T cells was measured by ELISA.Results:The transfection efficiency of plasmid was about 8%.There was significant difference between the cytolytic activity of T pcDNA3 1 MUC1/Y to SW620,Lovo and Raji.The cytolytic activity was about (42 8±6 15),(27 26±1 96)% and (22 73±2 15)% respectively.Compared with T IL 2)CTL induced by PBMC stimulated of IL 2(100 U/ml)) and T pcDNA3 1(CTL induced by DC transfected by pcDNA3 1+),the cytolytic activity of T pcDNA3 1 MUC1/Y against SW620 cell line showed a significant increase.The results of ANNEXIN V-FITC experimences showed that T pcDNA3 1 MUC1/Y could induce apoptosis of specific target cell.Conclusion:The expressing of MUC1/Y mRNA has important sense in gastrointestinal cancer.Constructed eukaryotic vector pIRES2 EGFP MUC1/Y that contains full length MUC1/Y cDNA could be used to study the transfection efficiency and select the positive clone;pcDAN3 1 MYC1/Y could induce powerful CTL immunoresponse.