闫智永,张爽,唐桥斐
YAN Zhiyong,ZHANG Shuang,TANG Qiaopei

• 1:沈阳医学院附属第二医院耳鼻喉科

摘要(Abstract)：

目的:探讨let-7e-5p对变应性鼻炎(AR)小鼠调节性T细胞(Treg)/辅助性T细胞17(Th17)失衡的影响。方法:12只BALB/c小鼠,3只为正常组,余9只小鼠采用卵清蛋白(OVA)和氢氧化铝[Al(OH)3]建立AR模型后随机均分为模型组(仅造模)、let-7e-5p激动剂对照组(鼻内滴注let-7e-5p agomir阴性对照)及let-7e-5p激动剂组(鼻内滴注let-7e-5p agomir);采用行为学评分考察各组小鼠的过敏症状,酶联免疫吸附法检测各组小鼠血清免疫球蛋白E(Ig E)、白细胞介素17-A(IL-17A)和白细胞介素-10(IL-10)水平,HE染色观察各组小鼠鼻黏膜组织学变化,采用实时荧光定量PCR检测各组小鼠鼻黏膜组织let-7e-5p的表达、蛋白印迹法检测RORγt和Foxp3蛋白的表达,采用流式细胞术检测各组小鼠外周血CD4+Foxp3+Treg和CD4+IL-17A+Th17细胞亚群变化。结果:模型组小鼠较正常组的行为学评分、血清Ig E及IL-17A水平明显增高、IL-10水平明显降低(P <0.01),let-7e-5p激动剂组较模型组的行为学评分、Ig E及IL-17A水平明显下降、IL-10水平明显升高(P <0. 01);同模型组比较,let-7e-5p激动剂组小鼠鼻黏膜基底层结构清晰,炎性细胞浸润明显减少;模型组小鼠鼻黏膜组织let-7e-5p基因的表达较正常组减少,let-7e-5p激动剂组则较模型组升高(P <0. 05);模型组小鼠鼻黏膜组织RORγt蛋白表达较正常组增加、Foxp3蛋白表达减少,let-7e-5p激动剂组小鼠鼻黏黏膜组织RORγt蛋白表达较模型组减少、Foxp3蛋白表达增加(P <0. 05);模型组小鼠较正常组外周血中CD4+Foxp3+Treg细胞亚群比例明显降低,CD4+IL-17A+Th17细胞亚群比例明显升高(P <0. 01); let-7e-5p激动剂组小鼠较模型组的CD4+Foxp3+Treg细胞亚群比例显著升高,CD4+IL-17A+Th17细胞亚群比例明显降低(P <0. 01)。结论:Let-7e-5p可能通过调节AR小鼠Foxp3和RORγt蛋白的表达,介导Treg/Th17细胞平衡,减轻炎症反应,从而改善AR小鼠鼻部过敏症状。
Objective: To investigate the effect of Let-7 e-5 p on imbalance of regulatory cells( Treg)/helper T cells( Th17) in mice with allergic rhinitis( AR). Methods: 12 BALB/c mice,3 mice in the normal group,and the remaining 9 mice were treated with ovalbumin( OVA) and aluminum hydroxide [Al( OH)3]to establish the AR model. They were randomly divided into the model group( for modeling only),the let-7 e-5 p agonist control group( intranasal infusion of let-7 e-5 p agomir negative control) and the let-7 e-5 p agonist group( intranasal infusion of let-7 e-5 p agomir). The behavioral score was used to investigate the allergic symptoms of the mice in each group. Serum Immunoglobulin E( IgE),IL-17 A and IL-10 levels in each group were detected by enzyme-linked immunosorbent assay. The pathological characteristics of the nasal mucosal tissues of the mice in each group were observed by HE staining. The let-7 e-5 p of the nasal mucosal tissues of the mice in each group was detected by real-time fluorescence quantitative PCR,and the expression of RORγt and Foxp3 in the nasal mucosal tissues of the mice in each group was detected by western blot. Changes in CD4+Foxp3+Treg and CD4+IL-17 A+Th17 subsets in peripheral blood of each group were detected by flow cytometry. Results: Compared with the normal group,the behavioral scores,serum IgE and IL-17 A levels of mice in the model group significantly increased,and IL-10 levels significantly decreased( P < 0. 01). Compared with the model group,the behavioral scores,IgE and IL-17 A levels of the let-7 e-5 p agonist group significantly decreased,and the IL-10 levels significantly increased( P <0. 01). Compared with the model group,the basal structure of the nasal mucosa tissues in the let-7 e-5 p agonist group was clear,and the infiltration of inflammatory cells was significantly reduced. The expression of let-7 e-5 p gene in the nasal mucosa of mice in the model group was decreased compared with that in the normal group. Compared with the normal group,the expression of RORγt protein increased and Foxp3 protein decreased in mouse nasal mucosa of model group. Compared with the model group,the expression of RORγt protein decreased and the expression of Foxp3 protein increased significantly in mouse nasal mucosa of let-7 e-5 p agonist group. The subsets of CD4+Foxp3+Treg cells in peripheral blood of mice in the model group were significantly lower than those in the normal group,and the subsets of CD4+IL-17 A+Th17 cells were significantly higher( P < 0. 01). The proportion of CD4+Foxp3+Treg subsets in the let-7 e-5 p agonist group was significantly higher than that in the model group,and the proportion of CD4+IL-17 A+Th17 subsets was significantly lower( P < 0. 01).Conclusion: let-7 e-5 p may mediate Treg/Th17 cell balance by regulating the expression of Foxp3 and RORγt proteins and reduce the inflammatory response,thereby improving the nasal allergy symptoms of AR mice.

关键词(KeyWords)： 小鼠;变应性鼻炎;let-7e-5p基因;辅助性T细胞17;调节性T细胞;1型辅助性T细胞;2型辅助T细胞
mice;allergic rhinitis(AR);let-7e-5p gene;T helper cell 17(Th17);regulatory T cells(Treg);T helper cells1(Th1);T helper cells2(Th2)

Abstract:

Keywords:

基金项目(Foundation): 辽宁省科学技术计划项目(2018225020);; 沈阳医学院科技基金项目(20182038)

作者(Author): 闫智永,张爽,唐桥斐
YAN Zhiyong,ZHANG Shuang,TANG Qiaopei

DOI: 10.19367/j.cnki.2096-8388.2020.05.008

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