MiR-146a在变应性鼻炎特异性免疫治疗中的增效作用

发布时间：2018-04-13 05:09

本文选题：变应性鼻炎 + 鼻黏膜　；参考：《山东大学》2016年博士论文

【摘要】：目的本研究为了探讨miR-146a在变应性鼻炎特异性免疫治疗中所发挥的作用及其相关作用机制,对其在变应性鼻炎患者鼻黏膜中的表达情况进行检测,随后通过建立变应性鼻炎小鼠模型并研究进行特异性免疫治疗时该小鼠模型中的miR-146a所发挥的作用,旨在为寻找新的特异性免疫增效因子并将之应用于临床提供实验依据。方法(1)荧光定量PCR法检测变应性患者以及非变应性鼻炎人群鼻黏膜中miR-146a mRNA的表达。(2)基础致敏阶段：以BALB/c小鼠为对象,依照50μg卵清蛋白(Ovalbumin, OVA)、5mg氢氧化铝凝胶为佐剂,加入1 mL灭菌生理盐水配制成悬浊液,随后对BALB/c小鼠进行腹腔注射,注射时间为隔日1次,总共注射7次；强化致敏阶段：将小鼠进行基础致敏之后,用5%OVA灭菌生理盐水溶液对各组实验小鼠进行滴鼻,每侧用量为10 μL,频率为每日1次,以此进行鼻腔激发,对AR模型组分别连续激发10天、20天,对照组小鼠依照上述相同方法用0.9%灭菌生理盐水进行腹腔注射,随后再用0.9%灭菌生理盐水进行滴鼻。酶联免疫法(ELISA)检测各组小鼠外周血OVA-sIgE含量,HE染色法观察各组小鼠鼻黏膜组织形态学变化情况,用荧光定量PCR检测各组小鼠鼻黏膜中miR-146a mRNA的表达情况。(3)分别用OVA (NPVO)、 miR-146a (NPVm)和OVA+miR-146a (NPVom)纳米颗粒疫苗作用于变应性鼻炎小鼠模型,观察进行上述操作后小鼠症状和相关指标的的变化情况。结果(1) miR-146a在变应性鼻炎患者鼻黏膜组织中的相对表达量明显低于非变应性鼻炎患者鼻黏膜组织(P0.01)；(2)造模结束后对小鼠鼻黏膜观察结果表明,AR模型组小鼠均具有典型的变应性鼻炎临床症状,其鼻黏膜上皮结构紊乱,炎性细胞浸润明显,纤毛部分融合甚至脱落；血清OVA-sIgE的浓度明显高于对照组(P0.05), miR-146a在AR模型组小鼠的鼻黏膜组织中的相对表达量明显低于正常鼻黏膜组织(P0.01)。(3) NPVo组小鼠的变应性鼻炎症状明显加重；NPVm处理组小鼠的变应性鼻炎相关反应无显著变化；NPVom处理组小鼠对变影响鼻炎相关反应表现出了明显的抑制作用；ELISA检测结果表明,各组小鼠的血清以及鼻黏膜中的sIgE以及IL-4水平亦明显增高；流式细胞术检测结果发现,OVA特异性抗原调节性T细胞的增殖活性显著上升；各组AR模型小鼠鼻黏膜以及脾脏中调节性T细胞的增殖活性明显低于正常对照组；分别用NPVo和NPVm处理的AR小鼠,其调节性T细胞增殖活性并未发生明显变化,而当用NPVom作用小鼠后,该组小鼠的T细胞增殖活性发生明显增高；进一步分析发现,NPVom处理组小鼠的调节性T细胞具有OVA特异性；NPVm或者NPVom作用小鼠后,能够使DCs中TGF-p mRNA及其相应蛋白的表达显著增高；用naive CD4+T细胞与NPV致敏的DCs进行共培养,结果表明NPVm/NPVom能够显著使naive CD4+CD25-T细胞转变为CD4+CD25+Foxp3+TGF+β+调节性T细胞；在用OVA作用后,由NPVom诱导的调节性T细胞的增殖活性显著增高,而由NPVm诱导的调节性T细胞的增殖活性则无明显变化；用牛血清白蛋白(BSA)作用于NPVom致敏DCs的增值情况进行检测,结果表明调节性T细胞的增殖活性无显著影响；分别将由NPVom诱导的调节性T细胞以及由NPVm诱导的调节性T细胞通过过继性转移的方式作用于变应性鼻炎小鼠,结果表明,由NPVom诱导的调节性T细胞能够明显抑制变应性鼻炎相关症状的出现,而由NPVm诱导的调节性T细胞或者naive CD4+T细胞则对变应性鼻炎相关症状无明显抑制作用。结论miR-146a能够通过诱导抗原特异性调节性T细胞(Treg)所介导的OVA特异性免疫治疗,促进Treg增殖分化,抑制抗原特异性免疫反应,从而促进变应性鼻炎的免疫治疗。
[Abstract]:Objective To investigate the role of miR - 14in allergic rhinitis specific immunotherapy and its related mechanism , and to detect the expression of miR - 14mRNA in the nasal mucosa of allergic rhinitis patients .
The results showed that ( 1 ) The relative expression of miR - 14s in nasal mucosa of allergic rhinitis patients was significantly lower than that in non - allergic rhinitis patients ( P0.01 ) .
( 2 ) The results of nasal mucosa observation showed that the AR model group had typical allergic rhinitis symptoms , its nasal mucosa epithelial structure disturbance , inflammatory cell infiltration and ciliate partial fusion or even shedding ;
The serum OVA - sIgE levels were significantly higher than those in the control group ( P0.05 ) , and the relative expression of miR - 14s in the nasal mucosa tissues of the AR model group was significantly lower than that in the normal nasal mucosa ( P0.01 ) .
There were no significant changes in allergic rhinitis - related reactions in mice treated with NPVm .
The effects of NPVom treatment group on allergic rhinitis were inhibited obviously .
The results of ELISA showed that the levels of sIgE and IL - 4 in serum and nasal mucosa of each group were also significantly increased .
The results of flow cytometry showed that the proliferation activity of OVA - specific antigen - regulatory T cells increased significantly ;
The proliferative activity of T cells in nasal mucosa and spleen of AR model mice was significantly lower than that of normal control group .
In AR mice treated with NPVo and NPVm , the proliferative activity of T cells was not changed significantly , but the proliferation activity of T cells increased significantly after NPVom was used in mice .
Furthermore , it was found that the regulatory T cells of NPVom treated group had OVA specificity .
After NPVm or NPVom , the expression of TGF - p mRNA and its corresponding protein in DCs was significantly increased .
The results showed that NPVm / NPVom could significantly transform naive CD4 + CD25 - T cells into CD4 + CD25 + Foxp3 + TGF + 尾 + regulatory T cells .
The proliferative activity of regulatory T cells induced by NPVom increased significantly after OVA , while the proliferative activity of regulatory T cells induced by NPVm did not change significantly ;
Bovine serum albumin ( BSA ) was used to detect the value - added of NPVom - sensitized DCs . The results showed that the proliferative activity of the regulatory T cells was not significantly affected .
Conclusion : The regulatory T cells induced by NPVom and regulatory T cells induced by NPVm can obviously inhibit the occurrence of allergic rhinitis , but the regulatory T cells or naive CD4 + T cells induced by NPVm can obviously inhibit the symptoms associated with allergic rhinitis .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R765.21

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