MUC1-2VNTR基因修饰的DC对骨髓瘤细胞的体外杀伤效应的研究

发布时间：2018-07-05 05:36

本文选题：多发性骨髓瘤 + 树突细胞　；参考：《昆明医科大学》2016年硕士论文

【摘要】：目的：通过体外杀伤实验,研究转染粘蛋白核心肽MUC1-2VNTR(两串联)基因的树突细胞(DC)诱导的细胞毒T细胞(CTL)对人的多发性骨髓瘤细胞株(U266)的诱导杀伤效应,为研制多发生性骨髓瘤基因疫苗奠定基础。方法：1、根据靶基因设计,将MUC1-2VNTR的编码基因作为目的基因,将人工合成的全基因克隆入载体pcDNA3.1/myc-hisA中。2、用去内毒素质粒小提试剂盒扩增、提取质粒、并保存。3、选取健康人的外周血单个核细胞,体外由重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和重组人白细胞介素4(rhIL-4)诱导成熟的DC细胞。4、构建pcDNA3.1-2VNTR重组质粒后,采用Lipofectimine脂质体介导法转染DC细胞(DC-2VNTR),以转染空载体质粒pcDNA3.1 (DC-pcDNA3.1)及lipofctamine处理的DC (DC-Lipo)作为对照组。5、Western印迹法检测重组体在DC细胞内的表达。6、在体外刺激同源T细胞,细胞毒实验检测DC-2VNTR诱导的CTL对多发性骨髓瘤细胞株(U266)的杀伤作用。结果：1.成功构建了重组质粒载体pcDNA3.1-2VNTR/myc-hisA;2.人的外周血DC细胞的培养和鉴定；3. Western印迹法检测到MUC1-2VNTR在DC细胞内的表达；4.DC-2VNTR诱导的CTL对多发性骨髓瘤细胞(U266)有显著杀伤效应,且显著高于DC-pcDNA3.1组及DC-Lipo组(P0.05)。结论：以MUC1-2VNTR基因转染的DC可以诱导CTL反应,并能特异地杀伤U266多发性骨髓瘤细胞株。
[Abstract]:Objective: to study the cytotoxic T cells (CTL) induced by dendritic cells (DC) transfected with MUC1-2VNTR gene in vitro on human multiple myeloma cell line (U266). To lay a foundation for the development of multiple myeloma gene vaccine. Methods according to the target gene design, the encoding gene of MUC1-2VNTR was used as the target gene, and the synthetic gene was cloned into the vector pcDNA3.1 / myc-hisA. In vitro, recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and recombinant human interleukin-4 (rhIL-4) were used to induce mature DC cells. The recombinant plasmid pcDNA3.1-2VNTR was constructed. DC cells were transfected with Lipofectimine liposome mediated transfection of DC cells (DC-2VNTR) and transfected with unloaded pcDNA3.1 (DC-pcDNA3.1) and lipofctamine treated DC (DC-Lipo) as control group. The expression of recombinant cells in DC cells was detected by Western blotting, and the homologous T cells were stimulated in vitro. Cytotoxicity assay was used to detect the cytotoxicity of CTL induced by DC-2 VNTR on multiple myeloma cell line (U266). The result is 1: 1. The recombinant plasmid pcDNA3.1-2VNTR / myc-hisA2 was successfully constructed. Culture and identification of human peripheral blood DC cells. The expression of MUC1-2VNTR in DC cells was detected by Western blot. CTL induced by DC-2VNTR had a significant killing effect on multiple myeloma cells (U266) and was significantly higher than that in DC-pcDNA3.1 group and DC-Lipo group (P0.05). Conclusion: DC transfected with MUC1-2VNTR gene can induce CTL response and specifically kill U266 multiple myeloma cell line.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.3

【相似文献】