摘要
目的:采用RNA干扰技术抑制小鼠骨髓树突状细胞(DC)髓样分化因子88(MyD88)的表达,并检测其对细胞生物学活性的影响,为DC的临床应用奠定基础。方法:针对MyD88基因,采用化学合成法合成3对MyD88 siRNA(序列1、序列2及序列3),并转染DC2.4细胞。采用半定量RT-PCR及Western blot分别从mRNA和蛋白质水平检测DCMyD88的表达情况,筛选其中一对高效RNA(序列3)转染DC2.4细胞(RNA干扰组),以未转染RNA的DC2.4细胞作为对照组,分别给予1 mg/L的脂多糖(LPS)刺激。流式细胞术(FCM)检测DC细胞表面CD80、CD86及MHC-Ⅱ分子的变化,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,免疫细胞化学检测DC核因子-κB(NF-κB)的表达,混合淋巴细胞培养检测T细胞增殖能力,观察RNA干扰对LPS促DC成熟的影响。结果:与空白对照组相比,序列2组及序列3组DC的mRNA和蛋白质表达分别降低90%和85%、92%和88%,差异有统计学意义;脂质体对照组、无义siRNA对照组及序列1组DC的mRNA和蛋白质表达无显著差异。经LPS刺激后,与对照组相比,RNA干扰组CD80、CD86及MHC-Ⅱ分子的表达,TNF-α、IFN-γ及IL-12的浓度及T细胞增殖能力均显著下降,且未见明显NF-κB核转位。结论:RNA干扰技术能显著下调小鼠DC MyD88的表达,并显著抑制LPS促DC成熟的效应,为以DC MyD88为靶向、相关疾病的基因治疗提供了新思路和手段。
AIM:To investigate inhibitory effects of RNA interference on MyD88 expression in murine myeloid dendritic cells(DCs)and detect the biological activity of DCs.METHODS:Three pairs of myeloid differentiation factor 88(MyD88)siRNA were synthesized and transfected into DCs by RNAi-mate.The mRNA and protein expression of MyD88 were analyzed by semi-quantified RT-PCR and Western blot.Mouse DCs were divided into control group and RNA interference group.One of the highest effective siRNA was transfected into RNA interference group.12 hours later,LPS of the final concentrations of 1.0 mg/L was added in two groups and continued to culture for 3 days.The phenotype and functional properties of DCs were detected by flow cytometry and mixed lymphocyte reaction(MLR).The concentration of TNF-α,IFN-y and IL-12 in the supernatant was detected by ELISA,and the concentration of NF-KB was detected by immunochemistry.RESULTS:mRNA and protein expression were reduced 90%and 85%in sequence2 siRNA,92%and 88%in sequence3 siRNA respectively but no change was found in other groups.LPS stimulation increased the expression of CD80,CD86 and MHC-Ⅱin the cytomembrane of DCs,the concentration of TNF-α,IFN-γand IL-12 in the supernatant in control group.Besides,LPS stimulation promoted the shift of NF-κB to karyon and the proliferation of allogeneic T cells in control group.RNA interference inhibited these effects induced by LPS,CONCLUSION:RNA interference can reduce MyD88 expression in murine myeloid dendritic cells and inhibit the maturation of DCs,which may provide a new strategy of gene therapy for related diseases.
作者
陈家军
孙宗全
董念国
苏刚
刘超
刘金平
邓勇志
CHEN Jia-jun;SUNZong-quan;DONG Nian-guo;SU Gang;LIU Chao;LIU Jin-ping;DENG Yong-zhi(Department of Cardiovascular Surgery,Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2007年第3期193-196,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(30471715)
