慢病毒载体介导的小鼠骨髓树突状细胞RelB基因沉默

目的: 利用慢病毒载体沉默小鼠骨髓树突状细胞(DC)的RelB基因, 建立RelB基因低表达的骨髓致耐受DC, 为自身免疫病的防治提供新方法.方法: 设计小鼠RelB基因干扰的靶序列R5, 合成并克隆到慢病毒载体上, 与慢病毒的包装材料在293FT细胞中包装成病毒颗粒, 收集病毒上清, 测定病毒滴度, 然后包装慢病毒感染DC, RT-PCR和免疫荧光方法检测, 灰度扫描并用软件分析RelB基因的表达水平, 未成熟DC作对照组, 选择T6包装病毒为RNAi的阴性对照.结果: RT-PCR和免疫荧光方法发现R5病毒感染的DC RelB基因表达水平与未成熟DC基因表达水平接近, 低于成熟DC的表达水平(P<0.05), 而实验对照T6组病毒感染的DC其RelB基因表达水平高于未成熟DC(P<0.05)和R5病毒转染DC组(P<0.05).结论: 小鼠骨髓DC表达的RelB基因被慢病毒载体有效沉默, 有望成为DC免疫治疗的新载体.

RelB silencing in mouse bone-marrow derived dendritic cells mediated by lentiviral vector

AIM: To silence RelB gene in mouse bone-marrow derived dendritic cells (DC) utilizing lentiviral vector, a novel tolerogenic dendritic cell with a relatively low expression level RelB was constructed and a new way to treat and prevent autoimmune diseases was explored. METHODS: Interferential targeting sequence R5 of RelB in mice was designed, synthesized and cloned into lentiviral vectors. Together with viral packaging materials were co-cultured in 293FT cell line to package lentiviral vector. Supernatant fluids were harvested, then virus titer detected. Mouse bone marrow derived DCs were infected by lentivirus particle. RelB gene expression level was detected by RT-PCR and immunofluorescence staining and analyzed by software of geo pro. There are three experiment control groups including immature DC, mature DC and DC infected by a negative independent control of T6. RESULTS: A similar RelB expression was detected by RT-PCR and immunofluorescence staining assay between DC infected virus R5 and immature DC, but was lower than that of mature DC. Significant difference in statisticsèP<0.05é. A similar RelB expression was detected by RT-PCR and immunofluorescence staining approachs between DC infected virus T6 and mature DC, but was higher than that of immature DC. Significant difference in statisticsèP<0.05é. CONCLUSION: RelB gene expressed by mouse bone marrow derived DC was silenced by Lentivirus vector effectively. The lentivirus vector with a low immunogenicity can be used to immunotherapy in vivo and overcome difficult transfection problem of primary DC. A new viral vector of DC immunotherapy can be obtained.