摘要
目的探讨RelB shRNA慢病毒感染对小鼠骨髓树突细胞(DC)的生物免疫学功能的影响。方法构建小鼠RelB shR-NA慢病毒载体。取小鼠股骨和胫骨的骨髓,采用重组粒-巨噬细胞集落刺激因子(rmGM-CSF)和重组白细胞介素4(rmIL-4)联合诱导小鼠骨髓DC。实验共分两组:对照组(小鼠骨髓未成熟DC组)和慢病毒感染组(RelBshRNA慢病毒感染的小鼠骨髓DC组)。两组DC均培养6d,经小鼠CD11c+免疫磁珠纯化,采用流式细胞仪检测DC表面MHC-Ⅱ、CD86和CD40的表达。取小鼠脾脏,收集T细胞。将两组DC与T细胞混合,按不同比例(1∶40、1∶20、1∶10和1∶5)设4个亚组,用MTT法检测T细胞增殖,细胞因子液相芯片联合试剂盒检测Th1类细胞因子(IL-2和IFN-γ)和Th2类细胞因子(IL-4和IL-10)的表达。结果RelB shRNA慢病毒感染DC的表面分子MHC-Ⅱ、CD86和CD40均呈低水平表达,其刺激异基因小鼠T细胞增殖的能力,以及Th1、Th2类细胞因子分泌水平与未成熟DC相当。结论RelB shRNA慢病毒感染的小鼠骨髓DC呈现出未成熟DC的表型和免疫功能。
Objective To explore the effect of RelB shRNA lentivirus infection on immunological function of bone marrow derived dendritic cells （DC） in mice Methods Mouse RelB shRNA lentiviral vector was constructed. Mouse bone marrow was collected from femur and tibia and cultured with recombinant mouse granulocyte-maerophage colony-stimulating factor （rmGM-CSF） and interleukin-4 （IL -4）. Two groups were set up.. control EC group （immature DC） and RNAi RelB DC group （DC infected by Lentivirus）, both groups were cultured for 6 days and then purified by mouse CD11c＋irnmunomagnetic bead. The expression levels of MHC-Ⅱ, CD86 and CD40 on the surface of DCs in two groups were detected by flow eytometry （FCM）. Allogeneic mouse T cells were isolated from spleen, DCs and T cells were co-cultured, and four subgroups were set up according to different reaction ratio （1：40, 1：20, 1：10 and 1：5）. T cells proliferation capacity was assessed detected by MTT approach. The expression levels of Thl （IL-2 and IFN-γ） and Th2 cytokines （IL-4 and IL-10） were determined by liquid chip kits method. Results The expression levels of co-stimulatory molecules （CD86 and CD40） and MHG-Ⅱ class molecule were low in DCs infected by RelB shRNA lentivirus. The DCs showed the same ability as the immature DCs in stimulating T-cell proliferation and Th1/Th2 secretion. Conclusion DC infected by RelB shRNA lentivirus has the same phenotype and immunological function as the immature DCs. RNAi RelB DC will be a potentially useful immunological agent in the treatment of many autoimmune diseases.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2009年第9期1127-1130,共4页
Medical Journal of Chinese People's Liberation Army
基金
广东省自然科学基金资助项目(8451051501000580)
