CD23-IgE介导跨上皮转运在变应性鼻炎发病机制中的作用

发布时间：2018-03-31 16:34

本文选题：CD23　切入点：IgE　出处：《复旦大学》2013年博士论文

【摘要】：第—部分增强的CD23-IgE介导大分子变应原跨上皮转运参与变应性鼻炎发病研究背景：由于上皮细胞之间的紧密连接的存在,阻碍大分子变应原(水溶性蛋白)自由通过上皮细胞层,大分子变应原如何通过鼻黏膜上皮屏障和黏膜固有层免疫效应细胞接触的机制尚不清楚。一些研究表明,IgE的低亲和力受体FcεRⅡ (CD23),具有转运IgE和IgE变应原免疫复合物通过极化的人单层消化道上皮的潜在功能；进一步研究发现,增强的CD23介导的跨上皮转运是启动消化道快速变应性炎症必不可少的步骤。但是人类鼻黏膜上皮是否存在同样的跨上皮转运IgE和IgE免疫复合物机制尚不清楚。目的：本课题第一部分拟在蛋白及mRNA水平检测变应性鼻炎患者和正常对照患者鼻黏膜上皮CD23的表达情况,并在体外研究人鼻黏膜上皮细胞表达的CD23是否具有转运IgE和IgE免疫复合物通过原代培养的人鼻上皮细胞单层的功能。材料和方法：采集20名我院行鼻内镜手术的鼻中隔偏曲并变应性鼻炎患者的术中矫正下鼻甲黏膜组织标本,经鼻内镜脑脊液鼻漏修补和颅底良性肿瘤切除术患者鼻腔黏膜作为对照。应用免疫荧光和Western Blot检测CD23在人鼻腔黏膜组织的表达：应用免疫荧光双标法检测CD23-IgE在鼻黏膜上皮细胞层结合位点；应用原位杂交的方法检测人鼻黏膜组织CD23a、CD23b mRNA表达情况。原代培养人鼻黏膜上皮细胞,通过广谱角蛋白的表达鉴定细胞的上皮源性,应用免疫荧光和Western Blot检测CD23在原代培养鼻黏膜上皮细胞的表达。于transwell透性支持物原代培养鼻黏膜上皮细胞,形成上皮细胞单层,通过检测跨上皮电阻和紧密连接ZO-1蛋白,确定单层细胞间紧密连接形成。应用透性支持物上的上皮细胞单层,模拟鼻黏膜上皮屏障,检测IgE在鼻黏膜上皮细胞单层顶面和底面之间的双向转运,和IgE抗原复合物从鼻黏膜上皮顶面(鼻腔面)至底面单向转运,并阻断CD23和IgE结合,说明上述转运是由CD23介导的,同时比较两钟不同源性的上皮细胞细胞单层转运的差异。结果：免疫荧光和Western Blot示CD23蛋白结构性的表达于人鼻黏膜上皮细胞,在过敏状态下其表达发生了上调；免疫荧光双标法示CD23-IgE的结合位点可位于鼻黏膜纤毛柱状上皮细胞顶面和基底面区域的细胞膜和细胞浆,变应性鼻炎患者鼻黏膜组织切片可见大量结合点,对照患者鼻黏膜组织切片偶见；原位杂交染色显示CD23a、CD23b mRNA均表达于人鼻黏膜上皮细胞,变应性鼻炎患者染色强度明显高于对照患者。免疫细胞化学和Western Blot示CD23在原代的培养的鼻黏膜上皮细胞均有表达,原代培养变应性鼻炎患者鼻上皮细胞的表达强度高于正常对照患者。CD23可以介导人IgE在透性支持物上的上皮细胞单层顶面-底面,底面-顶面的双向转运,亦可介导人IgE抗原复合物从上皮细胞单层顶面转运至底面,阻断CD23与IgE结合这种跨上皮转运现象变弱,IgE和IgE抗原复合物通过原代培养变应性鼻炎患者鼻上皮细胞单层的水平高于正常对照患者。结论：人鼻黏膜上皮细胞表达IgE的低亲和力受体FcεRⅡ (CD23), CD23可以介导转运IgE和IgE免疫复合物通过鼻黏膜上皮屏障,变应性鼻炎患者的这种跨上皮转运增强。第二部分在动物模型中经鼻CD23抗体干预抑制变应性鼻炎炎症反应目的：我们第一部分实验在体外证实CD23可以介导转运IgE双向通过鼻黏膜上皮细胞单层,可以介导转运IgE变应原复合物从顶面(鼻腔面)至基底面通过鼻黏膜上皮细胞单层,变应性鼻炎患者这种跨上皮转运增强。然而在体内阻断CD23-IgE介导跨上皮转运,是否可以抑制变应性鼻炎的发生,需要我们做进一步探讨。本实验先行应用免疫组化和Western Blot检测CD23在变应性鼻炎动物模型鼻腔黏膜的表达,然后在体内阻断CD23-IgE介导跨上皮转运,观察其对变应性鼻炎的影响。材料和方法：6-8周龄的雌性BALB/c小鼠,用卵清蛋白腹腔注射致敏,经鼻滴入激发建立小鼠变应性鼻炎模型。应用免疫组化和Western Blot检测CD23在小鼠鼻腔黏膜的表达。经鼻滴入CD23抗体B3B4阻断CD23-IgE介导跨上皮转运,同时应用同种型抗体和经鼻类固醇激素作为治疗的阴性和阳性对照。经鼻干预后,我们通过评估变应性鼻炎症状和病理组织学改变观察干预效果；同时通过观察血清和鼻腔灌洗液OVA特异性IgE、LTC4、ECP和IL-4水平,鼻黏膜组织中ECP水平和脾细胞反应情况评估干预效果。结果：CD23结构性的表达于小鼠鼻黏膜上皮细胞,在过敏状态下其表达发生了上调；在体内,CD23在鼻腔黏膜上皮细胞的上调与小鼠血清IL-4水平呈正相关,小鼠鼻腔灌洗液中IgE水平与CD23在鼻腔黏膜上皮细胞表达的量亦呈正相关。经鼻吸入CD23抗体B3B4后,小鼠鼻部症状减轻；组织学检查发现,鼻腔黏膜下嗜酸性粒细胞浸润减少；ELISA检测结果发现血清及鼻腔灌洗液OVA特异性IgE、LTC4、ECP和IL-4水平降低；Western Blot检测发现鼻黏膜组织ECP水平同样降低；流式细胞术示B3B4经鼻吸入干预纠正了CD4+辅助性T细胞反应向Th2细胞表型的偏移。这些结果提示在体内经鼻吸入CD23抗体干预抑制变应性鼻炎小鼠的炎症反应。结论：我们的研究提示CD23介导的跨上皮转运IgE和IgE变应原免疫复合物在变应性鼻炎发病机制中起重要作用,鼻黏膜上皮表达的CD23可作为变应性鼻炎治疗的新靶点。
[Abstract]:Part enhanced CD23-IgE mediates the trans epithelial transport of macromolecular allergens in the pathogenesis of allergic rhinitis
Background: due to the tight junctions between epithelial cells, hinder macromolecular allergen (soluble protein) free through the epithelial cell layer, the mechanism of how molecular allergen through the nasal mucosa epithelial barrier and lamina propria of immune effector cell contact is not clear. Some studies suggest that low affinity receptor Fc epsilon R II IgE the (CD23), with the transport of IgE and IgE allergen immune complexes by potential functional polarization of human digestive tract epithelial monolayer; further studies showed that the enhanced CD23 mediated transepithelial transport is essential for the initiation of rapid steps of allergic inflammation of the digestive tract. But whether human nasal epithelium has the same transepithelial transport of IgE IgE and immune complex mechanism is unclear.
Objective: the aim of this study was on the expression of protein and mRNA levels of patients with allergic rhinitis and normal nasal mucosa of patients with epithelial CD23, epithelial cells in monolayer and in vitro expression of human nasal epithelial cells CD23 with translocation of IgE and IgE immune complexes by primary cultured human nasal function.
Materials and methods: collected 20 underwent nasal endoscopic surgery and nasal septum in patients with allergic rhinitis during correction of inferior turbinate mucosa tissues, endoscopic repair of cerebrospinal fluid rhinorrhea and nasal mucosa of benign skull base tumor resection patients as control. The expression by immunofluorescence and Western Blot detection of CD23 in human nasal mucosa the detection of CD23-IgE double immunofluorescence method in nasal mucosa epithelial cell layer binding sites; with the method of in situ hybridization CD23a detection of human nasal mucosa, CD23b. The expression of mRNA in primary cultured human nasal epithelial cells, epithelial cells were identified by expression of cytokeratin, immunofluorescence and Western Blot detection CD23 expressed in cultured nasal epithelial cells in primary Transwell. To support the permeability of primary cultured nasal epithelial cells, formation of epithelial cell monolayer, through Detection of transepithelial electrical resistance and tight junction protein ZO-1, determine the close connection between the cell monolayer permeability formation. The application supports the epithelial cell monolayer, simulated nasal epithelial barrier, the bidirectional transfer of detection of IgE in nasal mucosa epithelial cell monolayer between the top and bottom surfaces, and IgE antigen complex from the nasal epithelium top face (nasal surface) to the bottom surface of one-way transport, and block CD23 and IgE combination, the transport is mediated by CD23, and the differences between epithelial cell monolayer transport between the two different origin of the clock.
Results: immunofluorescence and Western Blot showed that the expression of CD23 protein structural in human nasal epithelial cells, its expression in allergic condition occurs raised; binding sites in CD23-IgE immunofluorescence were located in the epithelial cells of the nasal ciliated columnar and basal area of the top surface of the cell membrane and cytoplasm in allergic rhinitis the nasal mucosa tissue sections showed a large number of binding sites, the control sections of nasal mucosa of patients I see; in situ hybridization showed that CD23a, CD23b and mRNA were expressed in human nasal epithelial cells in patients with allergic rhinitis, the staining intensity was significantly higher than the control patients. Immunocytochemistry and Western Blot showed the expression of CD23 in nasal mucosa epithelial cells of primary culture generation, the expression intensity of primary cultured in patients with allergic rhinitis nasal epithelial cells than that of normal control patients with.CD23 can be mediated by IgE in permeability on a support The top surface of epithelial cell monolayer - bottom, bottom - top surface of the bidirectional transfer, can also be mediated by human IgE antigen complexes from epithelial cell monolayer transport to the top surface of bottom surface, blocking CD23 and IgE combined with the transepithelial transport of weak, IgE and IgE antigen in patients with allergic nasal nasal epithelial cells monolayer the level is higher than the normal control patients by primary culture.
Conclusion: human nasal epithelial cells express IgE low affinity receptor Fc epsilon R II (CD23). CD23 can mediate the transport of IgE and IgE immune complexes through the nasal epithelial barrier and enhance the cross epithelial transport in patients with allergic rhinitis.
The second part of the animal model with nasal CD23 antibody intervention to inhibit the inflammatory response of allergic rhinitis
Objective: the first part of our experiment in vitro showed that CD23 could be mediated through bidirectional transport of IgE nasal epithelial cells monolayer, can mediate the transport of IgE allergen complex from the top (nasal surface) to the basal surface of the epithelial cells of nasal mucosa of patients with allergic rhinitis in the monolayer, transepithelial transport is enhanced. However, in vivo blockade of CD23-IgE mediated the transepithelial transport, whether can inhibit the occurrence of allergic rhinitis, we need to do further study. The first experiment used immunohistochemistry to investigate the expression of Western and Blot detection of CD23 in nasal mucosa of allergic rhinitis animal model, and in vivo blocking CD23-IgE mediated transepithelial transport, to observe its effect on allergic rhinitis.
Materials and methods: 6-8 week old female BALB/c mice with intraperitoneal injection of ovalbumin sensitized by nasal instillation induced mouse model of allergic rhinitis. The expression of immunohistochemical detection of CD23 and Western Blot in nasal mucosa of mice. Intranasal instillation of CD23 B3B4 antibody blocking CD23-IgE mediated transepithelial transport, and application isotype antibody and nasal steroid treatment as negative and positive controls. The prognosis by evaluating our nose, allergic rhinitis symptoms and histopathological changes were observed at the same time through the intervention effect; observation of serum and nasal lavage fluid of OVA specific IgE, LTC4, ECP and IL-4 level, ECP level and spleen cell response in nasal mucosa in the evaluation of the intervention effect.
Results: CD23 structural expression in nasal mucosa epithelial cells in allergic mice, under the condition of its expression occurs raised; in vivo, CD23 was positively correlated in nasal mucosa epithelial cells and increased serum IL-4 levels, IgE levels and CD23 mice in the nasal lavage fluid in nasal mucosal epithelial cells is also positively related to the amount. By inhalation of CD23 antibody B3B4 mice after nasal symptoms relieved; histological examination revealed that the nasal submucosal eosinophilic acid granulocyte infiltration; ELISA test results showed that serum and nasal lavage fluid of OVA specific IgE, LTC4, ECP and IL-4 levels decreased; Western Blot detected ECP levels in nasal mucosa also showed B3B4 decreased; nasal inhalation intervention to correct the offset of CD4+ helper T cell responses to Th2 cell phenotype by flow cytometry. These results suggest that in vivo by inhalation of CD23 antibody inhibits allergic nasal Inflammation in the inflammatory mice.
Conclusion: our study suggests that CD23 mediated transepithelial transport of IgE and IgE allergen immune complexes plays an important role in the pathogenesis of allergic rhinitis. CD23 expressed on nasal mucosa can serve as a new target for treatment of allergic rhinitis.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R765.21

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