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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DCRelB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。
Objective: To silence RelB gene of mouse bone marrow dendritic cells (DC) by lentiviral vector and to establish low resistance bone marrow-derived DCs with RelB gene. This study provides a new method for the prevention and treatment of autoimmune diseases. Methods: The targeting sequence R5 of mouse RelB gene was designed, synthesized and cloned into the lentiviral vector. The lentiviral packaging material was packaged into virus particles in 293FT cells, the virus supernatant was collected, the virus titer was determined, and then packaged slowly Virus infection was detected by DC, RT-PCR and immunofluorescence method. The expression of RelB gene was analyzed by gray-scale scanning software. Immature DC as control group, T6 packaging virus was selected as the negative control of RNAi. Results: RT-PCR and immunofluorescence showed that the expression level of DCRelB gene in A549-infected mice was close to that in immature DCs, lower than that of mature DCs (P <0.05) RelB gene expression level was higher than immature DC (P <0.05) and R5 virus transfected DC group (P <0.05). CONCLUSION: RelB gene expressed in mouse bone marrow DCs is effectively silenced by lentiviral vector and is expected to become a new carrier of DC immunotherapy.