摘要
目的探讨siRNA沉默Rel-A基因表达对大鼠树突状细胞(DC)生物学效应的影响。方法采用细胞因子诱导培养法体外扩增大鼠骨髓DC,经免疫磁珠纯化获得高纯度DC后,针对Rel-A基因,采用半定量RT-PCR及Western blot分别从mRNA和蛋白质水平检测Rel-A的表达情况,筛选一对高效RNA干扰(RNAi)行慢病毒载体包装并转染DC(RNA干扰组),以未转染RNAi的DC作为对照组。分别给予1mg/L的脂多糖(LPS)刺激,透射电镜观察RNAi对LPS促DC成熟的影响,并利用流式细胞术(FCM)检测DC表面CD80,CD86及MHCⅡ分子的变化;混合淋巴细胞反应(MLR)法检测T细胞增殖能力;ELISA法检测DC分泌肿瘤坏死因子α(TNF-α)、干扰素γ(IFN-γ)和白介素12(IL-12)的浓度。结果与空白对照组相比,序列2组降低最明显,抑制率达90%,差异有统计学意义;透射电镜下可见RNAi Rel-A DC内多吞噬泡样结构;RNA干扰组CD80,CD86,MHCⅡ分子的表达、T细胞增殖能力及TNF-α,IFN-γ,IL-12的浓度与对照组相比均显著下降。结论RNA干扰技术能显著下调大鼠Rel-A基因的表达,Rel-A基因沉默DC的生物学效应表现为未成熟DC的特点。这种RNAi Rel-A DC可望作为一种新型的致耐受DC,而应用于免疫耐受的诱导研究。
Objective To study the biological effects of silencing of Rel-A gene expression by RNA interference on the dendritic ceils. Methods Mouse dendritic cells derived from bone marrow were amplificated through cytokine inducing system and purified with positive immunomicrobeads by flow cytometer. The mRNA and protein expression, aiming directly at Rel-A, were analyzed by semi-quantified RT-PCR and Western blot. A pair of the highest effective siRNA were synthesized selectively and packed by lentivirus transfer vector, then transfected to DC （RNA interference group ） , while control group was not transfected by RNAi ; 12 hours later, lipipolysaccharide （ LPS ） at a concentration of 1 mg/L was added to two groups and continued to culture. The cell microstructure was then observed under transmission electron microscopy. The cell surfacemarkers, such as MHC Ⅱ , CD86 and CD80, were detected by flow cytometer. The functional properties of DCs were detected by mixed lymphocyte reaction （ MLR ） . The concentration of TNF-α, IFN-γ and IL-12 in the supernatant were detected by ELISA. Results mRNA and protein expression were reduced 90% in sequence 2 siRNA, the difference was significant statistically, in comparison with control group. Many phagocytic vacuoles were observed in the cytoplasm under transmission electron microscopy. The expression of CD80, CD86 and MHC Ⅱ in the eytomembrane of DCs, the proliferation of allogeneie T cells and the concentration of TNF-α, IFN-γ and IL-12 decreased more significantly in RNA interference group than in control group. Conclusions RNA interference can reduce Rel-A expression in murine myeloid dendritic cells and inhibit the maturation of DCs. The dendritic cells interfered with silencing of Rel-A gene are characterized as the biological features and immunological function of immature DC. This kind of RNAi Rel-A DCs may be used to induce immunotolerance as novel tolerogenic DCs.
出处
《中国普通外科杂志》
CAS
CSCD
北大核心
2009年第1期44-48,共5页
China Journal of General Surgery
基金
江苏省自然科学基金资助项目(BK2007057)
江苏省普通高校研究生科研创新计划项目
