GATA-3小干扰RNA载体对变应性鼻炎小鼠外周血单个核细胞中辅助性T细胞亚群功能的体内调节机制  

In vivo regulatory effect of si-GATA-3 on the Th1/Th2 imbalance in murine allergic rhinitis models

作　　者：李玉晓[1,2] 贺铭 王天蓉 王雨 LI Yuxiao;HE Ming;WANG Tianrong;WANG Yu(Department of Otolarygology Head and Neck Surgery,the First Affliated Hospital of Kunming Medical University,Kunming,Yunnan,650032,China;Department of Otolaryngology,the Seventh Affiliated Hospital,Sun Yat-sen University,Shenzhen,Guangdong,517108,China;Department of Biochemistry and Molecular Biology,School of Basic Medical Science,Kunming MedicalUniversity,Kunming,Yunnan,650500,China)

机构地区：[1]昆明医科大学第一附属医院耳鼻咽喉头颈外科,云南昆明650032 [2]中山大学附属第七医院(深圳)耳鼻咽喉科,广东深圳517108 [3]昆明医科大学基础医学院,生物化学与分子生物学教研室,云南昆明650500

出　　处：《中国耳鼻咽喉头颈外科》2023年第10期646-651,共6页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：国家自然科学基金(81960185);云南省科技厅-昆明医科大学应用基础研究联合专项基金[2018FE001(-205)]。

摘　　要：目的 研究RNA干扰(RNA interference,RNAi)介导的GATA-3小干扰RNA载体(si-GATA-3)对变应性鼻炎(AR)小鼠外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)中辅助性T细胞1(T-helper 1,Th1)和Th2功能的调节作用。方法 构建si-GATA-3,用卵清蛋白构建BALB/c AR模型,si-GATA-3组、si-NC组(无目标基因小干扰RNA的空白对照组)分别用si-GATA-3和si-NC腹腔注射干预AR小鼠,AR组和正常对照组用等量的生理盐水干预。干预结束抽取其外周血,分离PBMCs和血浆。分别用荧光定量PCR和Western blot技术检测PBMCs中GATA-3和T-bet mRNA及其相应蛋白的表达量;用ELISA技术检测血浆中白细胞介素4(IL-4)和干扰素γ(IFN-γ)的含量。结果 si-GATA-3滴度为5×10^(8)TU/ml。成功制备BALB/C小鼠AR模型。si-GATA-3能明显改善AR小鼠的症状。si-GATA-3组PBMCs中GATA-3 mRNA表达明显低于AR组和si-NC组(P<0.01),AR组和si-NC组明显高于正常对照组(P<0.01)。PBMCs中GATA-3蛋白和血浆中IL-4的表达量与GATA-3 mRNA表达相似。si-GATA-3组PBMCs中T-bet mRNA及其蛋白的相对表达均明显高于AR组和si-NC组(P<0.01),AR组和si-NC组二者均明显低于对照组(P<0.01)。si-GATA-3组血浆中IFN-γ的表达量高于正常对照组(P<0.05);AR组与si-NC组中IFN-γ的表达量均明显高于正常对照组和si-GATA-3组(P<0.01)。结论 si-GATA-3能有效改善AR小鼠症状,下调AR小鼠PBMCs中Th2细胞中GATA-3 mRNA、GATA-3蛋白和IL-4的表达,同时上调Th1细胞中T-bet mRNA和T-bet蛋白的表达,纠正Th2/Th1的免疫失衡。OBJECTIVE To investigate in vivo regulatory effect of the lentivirus-mediated-GATA-3 RNAi(si-GATA-3)on the Th1/Th2 imbalance in murine allergic rhinitis models.METHODS Construction of si-GATA-3.BALB/c Murine AR models were established by ovalbumin(OVA).The mice in si-GATA-3 group were injected with si-GATA-3 intraperitoneally,and si-NC for si-NC mice.The mice in both the AR group and the control group were injected intraperitoneally with physical saline.The peripheral blood samples of mice were extracted,and the peripheral blood mononuclear cells(PBMCs)and serum were separated.The levels of IL-4 and IFN-γin serum were detected by ELISA.Q-PCR and Western blot analysis was used to determine the levels of T-bet and GATA-3 mRNA and proteins in PBMCs,respectively.RESULTS Concentration of siGATA-3 is 5×10^(8) TU/ml.The expression level of GATA-3 mRNA in si-GATA-3 group was lower than that in both si-NC group and AR group,respectively(P<0.01),and that in both si-NC group and AR group was higer than control group(P<0.01).The expression level of GATA-3 protein and IL-4 in serum in si-GATA-3 group were similar with those of GATA-3 mRNA.The expression levels of T-bet mRNA and protein in PBMCs in si-GATA-3 group were higer than those in both si-NC group and AR group,respectively(P<0.01),and the data showed significant difrerence in those in control group compared to si-GATA-3 group(P<0.05).The expression level of IFN-γin serum in si-GATA3 group was lower than that in both AR group and si-NC group,respectively(P<0.05).CONCLUSION Si-GATA-3 could down-regulate the expressions of mRNA and protein of GATA-3 and IL-4,and up-regulate the expression of mRNA and protein of T-bet and IFN-γeffectively in vivo.Therefore,it could correct the immunol imbalance of Th1/Th2 in the murine AR models.

关 键 词：鼻炎 过敏 RNA干扰 模型 动物 小鼠 GATA-3 T-BET 慢病毒载体 

分 类 号：R765.21[医药卫生—耳鼻咽喉科]