RNAi阻断GATA-3表达治疗小鼠过敏性鼻炎的实验研究  

Suppression of GATA-3 expression by RNAi in treatment of murine allergic rhinitis

作　　者：郑铭[1] 王妍[2] 周兵[1] 杜芝燕[2] 徐元基[2] 于晓妉[2] 

机构地区：[1]首都医科大学附属北京同仁医院耳鼻咽喉头颈外科耳鼻咽喉头颈科学教育部重点实验室,北京100730 [2]军事医学科学院基础医学研究所,北京100850

出　　处：《军事医学》2011年第12期896-901,共6页Military Medical Sciences

基　　金：国家自然科学基金资助项目(30471871);国家和北京市新世纪百千万人才工程培养经费个人资助项目(B级;2005.09-2008.10)

摘　　要：目的探讨以GATA-3为基因治疗靶标,在体内实验阶段评估RNAi技术抑制Th2极化反应,阻止过敏性鼻炎发展的潜在治疗作用。方法采用脂质体鼻腔给药系统,将在体外实验中获得的针对GATA-3基因抑制效果最明显的特异性siRNA重组质粒载体Si338应用于小鼠过敏性鼻炎模型上,借助RT-PCR、ELISA、流式细胞术和HE染色分别检测鼻腔黏膜GATA-3的mRNA水平和相关炎症因子变化,以及Th1/Th2细胞亚群漂移和炎症细胞浸润的改变。结果①HE染色后,显微镜下观察到过敏性鼻炎小鼠鼻黏膜水肿,上皮脱落,炎症细胞浸润增加和嗜酸性粒细胞数均高于正常对照组,经RNAi治疗后,鼻黏膜中嗜酸性粒细胞数明显下降。②RT-PCR显示s,iRNA质粒载体有效下调Th2极化条件下的GATA-3 mRNA含量。③流式细胞术检测显示,过敏性鼻炎小鼠脾脏中,Th1/Th2细胞亚群出现漂移,Th1细胞的相对数量明显减少,Th2细胞相对数量明显增加,RNAi治疗后,Th1/Th2比率出现逆转。④ELISA显示过敏性鼻炎小鼠外周血中I,L-4与IL-5含量增高,Si338组IL-4含量明显降低。结论在获得高效、特异性干涉序列基础上,针对疾病上游基因,诸如GATA-3,给予RNAi技术的靶向治疗能够显著缓解过敏性鼻炎动物模型中鼻黏膜的Ⅰ型变态反应。本研究为今后前瞻性地探索siRNA基因治疗过敏性鼻炎提供了具有潜力的研究思路和可行的实验方法。Objectives To investigate the effect of GATA-3 siRNA on Th2 differentiation and to evaluate the therapeutic potential of RNAi in allergic rhinitis in vivo.Methods siRNA plasmid-Si338 effective for and specific to the inhibition of GATA-3 gene expression obtained from in vitro experiments was used in the murine allergic rhinitis via nasal administration of liposomes.The extent of inflammatory infiltration in the nasal mucosa,expression of mucosal GATA-3 mRNA,shift of the percentage of Th1/Th2 and levels of plasma related cytokines were detected by stained hematoxylin-eosin,RT-PCR,flow cytometry and ELISA,separately.Results ①A microscope was used to reveal that the swelling of the nasal mucosa,the detachment of epithelia,and the increase of the infiltration of inflammatory cells and amount of neutrophils in model group compared with control group,while GATA-3 siRNA decreased the amount of neutrophils.②The experimental results of RT-PCT exhibited that the level of GATA-3 mRNA was decreased significantly after administration of Si338 plasmid.③ Flow cytometry was performed to indicate down-regulation of Th1 cells and up-regulation of Th2 cells in the spleen of allergic rhinitis mice.Furthermore,specific GATA-3 siRNA contributed to an elevated Th1/Th2 ratio involving the allergic response.④ ELISA demonstrated that IL-4 and IL-5 were elevated in allergic rhinitis mice,and IL-4 was significantly repressed in Si338 group.Conclusion This study suggests a therapeutic approach that targets upstream genes,such as GATA-3,through intranasal mucosal administration of target gene siRNA,can attenuate Th2 differentiation and alleviate the animal model with allergic rhinitis.Thus,RNAi-based gene therapy might provide a potentially efficient strategy particularly in Th2-mediated inflammatory diseases,such as allergic rhinitis.

关 键 词：GATA3转录因子 过敏性鼻炎 动物模型 RNA干扰 

分 类 号：R765.21[医药卫生—耳鼻咽喉科]