siRNA沉默STAT6抑制嗜酸粒细胞性慢性鼻窦炎产生的实验性研究  

Silencing STAT6 with siRNA prevents development of eosinophilic chronic rhinosinusitis:an experimental study

作　　者：卫红齐[1] 朱政文[1] 邢洪瑜 刘志勇[1] 曹忠胜[1] 徐龙江[2] 刘济生[3] Wei Hongqi;Zhu Zhengwen;Xing Hongyu;Liu Zhiyong;Cao Zhongsheng;Xu Longjiang;Liu Jisheng(Department of Otorhinolaryngology,the Second Affiliated Hospital of Soochow University,Suzhou 215004,China;Department of Pathology,the Second Affiliated Hospital of Soochow University,Suzhou 215004,China;Department of Otorhinolaryngology,the First Affiliated Hospital of Soochow University,Suzhou 215006,China)

机构地区：[1]苏州大学附属第二医院耳鼻咽喉科,苏州215004 [2]苏州大学附属第二医院病理科,苏州215004 [3]苏州大学附属第一医院耳鼻咽喉科,苏州215006

出　　处：《中华耳鼻咽喉头颈外科杂志》2023年第11期1102-1111,共10页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：苏州市科技计划项目(SKJY2021084)。

摘　　要：目的探讨用小干扰RNA(siRNA)沉默信号传导和转录激活因子6(signal transducer and activator of transcription 6,STAT6),能否抑制小鼠嗜酸粒细胞性慢性鼻窦炎(eosinophilic chronic rhinosinusitis,ECRS)的产生。方法2022年3—9月期间,48只BALB/c雌性小鼠随机分为Control(对照)组、Vehicle(转染试剂)组、Scramble siRNA(对照siRNA)组和STAT6 siRNA组,每组12只。用卵清蛋白(OVA)联合金黄色葡萄球菌肠毒素B(staphylococcal enterotoxin B,SEB)诱导小鼠ECRS,并用siRNA鼻腔滴注进行干预。随后收集外周血以及鼻黏膜组织标本。血细胞分析仪检测外周血嗜酸粒细胞(Eos)的数目及百分比;酶联免疫吸附实验检测外周血总免疫球蛋白E(IgE)和OVA-特异性IgE(OVA-sIgE)水平,以及鼻黏膜组织中干扰素γ(IFN-γ)、白细胞介素(IL)-5、IL-17A和嗜酸粒细胞趋化因子1(Eotaxin-1)的表达量;用苏木精-伊红(HE)染色鼻黏膜组织切片,在高倍视野(HPF)下对Eos进行计数;蛋白免疫印迹检测鼻黏膜中STAT6和磷酸化STAT6(p-STAT6)蛋白水平;免疫荧光染色对鼻黏膜中p-STAT6表达进行定位。采用SPSS 18.0软件进行统计学分析。结果STAT6 siRNA组小鼠外周血Eos计数、Eos百分比、总IgE以及OVA-sIgE水平[(0.318±0.045)×10^(3)/μl、(3.667±0.479)%、(102.070±13.205)ng/ml和(38.870±7.352)ng/ml]明显低于Vehicle组[(0.532±0.049)×10^(3)/μl、(6.710±1.061)%、(203.102±29.653)ng/ml和(74.575±6.432)ng/ml,Z值分别为-2.56、-2.24、-2.40、-2.56,P值均<0.05]和Scramble siRNA组[(0.493±0.036)×10^(3)/μl、(5.858±0.872)%、(189.964±30.042)ng/ml和(80.935±8.358)ng/ml,Z值分别为-2.17、-2.08、-2.24、-2.72,P值均<0.05],且鼻黏膜组织中IL-5和Eotaxin-1表达量分别为(312.279±34.281)pg/ml和(25.297±4.323)pg/ml,也显著低于Vehicle组[(689.667±31.905)pg/ml、(68.278±6.485)pg/ml,Z值分别为-2.73、-2.88,P值均<0.01]和Scramble siRNA组[(661.783±42.094)pg/ml、(63.015±7.416)pg/ml,Z值分别为-2.72、-2.81,P值均<0.01]。STAT6 siObjective To investigate whether silencing signal transducer and activator of transcription 6(STAT6)with siRNA can inhibit eosinophilic inflammation of sinonasal mucosa in a mouse model of eosinophilic chronic rhinosinusitis(ECRS).Methods The study was conducted from March to September in 2022.Forty-eight female BALB/c mice were randomly divided into four groups:the control group,the Vehicle(transfection reagent)-treated group,the Scramble siRNA(Control siRNA)-treated group,and the STAT6 siRNA-treated group,with twelve mice in each group.An ovalbumin(OVA)-staphylococcal enterotoxin B(SEB)-induced ECRS murine model was established.SiRNA prepared in Lipofectamine was locally administered to the nasal cavity.After administration,samples of the peripheral blood and sinonasal mucosa were collected.Eosinophils in peripheral blood were detected by hematology analyzer.Total and OVA-specific IgE(OVA-sIgE)in serum were detected by enzyme-linked immunosorbent assay(ELISA).Mucosal levels of cytokines and chemokines,including interleukin(IL)-5,IL-17A,interferon-γ(IFN-γ)and eotaxin-1,were also measured using ELISA.Mucosal histological changes of eosinophil infiltration were examined using hematoxylin,and eosin staining,and tissue eosinophil count was performed using a microscope under a high-power field(HPF).Tissue expression of STAT6 and phosphorylated STAT6(p-STAT6)was detected with the western blot method.Immunofluorescence staining was used to localize the expression of p-STAT6 in sinonasal mucosa.Statistical analysis was conducted using SPSS 18.0 software.Results Peripheral blood eosinophil count,percentage of peripheral blood eosinophil,total serum IgE level,and serum OVA-sIgE level in the STAT6 siRNA-treated group[(0.318±0.045)×10^(3)/μl,(3.667±0.479)%,(102.070±13.205)ng/ml,and(38.870±7.352)ng/ml]were significantly different from those of the Vehicle-treated group[(0.532±0.049)×10^(3)/μl,(6.710±1.061)%,(203.102±29.653)ng/ml,and(74.575±6.432)ng/ml,Z value was-2.56,-2.24,-2.40,and-2.56,respectively,all P<0.0

关 键 词：鼻窦炎 嗜酸粒细胞 Th2免疫反应 信号传导和转录激活因子6 小干扰RNA 

分 类 号：R765.41[医药卫生—耳鼻咽喉科]