本文選題：CD23　切入點：IgE　出處：《復旦大學》2013年博士論文

【摘要】：第—部分增強的CD23-IgE介導大分子變應原跨上皮轉運參與變應性鼻炎發(fā)病 研究背景：由于上皮細胞之間的緊密連接的存在,阻礙大分子變應原(水溶性蛋白)自由通過上皮細胞層,大分子變應原如何通過鼻黏膜上皮屏障和黏膜固有層免疫效應細胞接觸的機制尚不清楚。一些研究表明,IgE的低親和力受體FcεRⅡ (CD23),具有轉運IgE和IgE變應原免疫復合物通過極化的人單層消化道上皮的潛在功能；進一步研究發(fā)現(xiàn),增強的CD23介導的跨上皮轉運是啟動消化道快速變應性炎癥必不可少的步驟。但是人類鼻黏膜上皮是否存在同樣的跨上皮轉運IgE和IgE免疫復合物機制尚不清楚。 目的：本課題第一部分擬在蛋白及mRNA水平檢測變應性鼻炎患者和正常對照患者鼻黏膜上皮CD23的表達情況,并在體外研究人鼻黏膜上皮細胞表達的CD23是否具有轉運IgE和IgE免疫復合物通過原代培養(yǎng)的人鼻上皮細胞單層的功能。 材料和方法：采集20名我院行鼻內(nèi)鏡手術的鼻中隔偏曲并變應性鼻炎患者的術中矯正下鼻甲黏膜組織標本,經(jīng)鼻內(nèi)鏡腦脊液鼻漏修補和顱底良性腫瘤切除術患者鼻腔黏膜作為對照。應用免疫熒光和Western Blot檢測CD23在人鼻腔黏膜組織的表達：應用免疫熒光雙標法檢測CD23-IgE在鼻黏膜上皮細胞層結合位點；應用原位雜交的方法檢測人鼻黏膜組織CD23a、CD23b mRNA表達情況。原代培養(yǎng)人鼻黏膜上皮細胞,通過廣譜角蛋白的表達鑒定細胞的上皮源性,應用免疫熒光和Western Blot檢測CD23在原代培養(yǎng)鼻黏膜上皮細胞的表達。于transwell透性支持物原代培養(yǎng)鼻黏膜上皮細胞,形成上皮細胞單層,通過檢測跨上皮電阻和緊密連接ZO-1蛋白,確定單層細胞間緊密連接形成。應用透性支持物上的上皮細胞單層,模擬鼻黏膜上皮屏障,檢測IgE在鼻黏膜上皮細胞單層頂面和底面之間的雙向轉運,和IgE抗原復合物從鼻黏膜上皮頂面(鼻腔面)至底面單向轉運,并阻斷CD23和IgE結合,說明上述轉運是由CD23介導的,同時比較兩鐘不同源性的上皮細胞細胞單層轉運的差異。 結果：免疫熒光和Western Blot示CD23蛋白結構性的表達于人鼻黏膜上皮細胞,在過敏狀態(tài)下其表達發(fā)生了上調；免疫熒光雙標法示CD23-IgE的結合位點可位于鼻黏膜纖毛柱狀上皮細胞頂面和基底面區(qū)域的細胞膜和細胞漿,變應性鼻炎患者鼻黏膜組織切片可見大量結合點,對照患者鼻黏膜組織切片偶見；原位雜交染色顯示CD23a、CD23b mRNA均表達于人鼻黏膜上皮細胞,變應性鼻炎患者染色強度明顯高于對照患者。免疫細胞化學和Western Blot示CD23在原代的培養(yǎng)的鼻黏膜上皮細胞均有表達,原代培養(yǎng)變應性鼻炎患者鼻上皮細胞的表達強度高于正常對照患者。CD23可以介導人IgE在透性支持物上的上皮細胞單層頂面-底面,底面-頂面的雙向轉運,亦可介導人IgE抗原復合物從上皮細胞單層頂面轉運至底面,阻斷CD23與IgE結合這種跨上皮轉運現(xiàn)象變?nèi)?IgE和IgE抗原復合物通過原代培養(yǎng)變應性鼻炎患者鼻上皮細胞單層的水平高于正常對照患者。 結論：人鼻黏膜上皮細胞表達IgE的低親和力受體FcεRⅡ (CD23), CD23可以介導轉運IgE和IgE免疫復合物通過鼻黏膜上皮屏障,變應性鼻炎患者的這種跨上皮轉運增強。 第二部分在動物模型中經(jīng)鼻CD23抗體干預抑制變應性鼻炎炎癥反應 目的：我們第一部分實驗在體外證實CD23可以介導轉運IgE雙向通過鼻黏膜上皮細胞單層,可以介導轉運IgE變應原復合物從頂面(鼻腔面)至基底面通過鼻黏膜上皮細胞單層,變應性鼻炎患者這種跨上皮轉運增強。然而在體內(nèi)阻斷CD23-IgE介導跨上皮轉運,是否可以抑制變應性鼻炎的發(fā)生,需要我們做進一步探討。本實驗先行應用免疫組化和Western Blot檢測CD23在變應性鼻炎動物模型鼻腔黏膜的表達,然后在體內(nèi)阻斷CD23-IgE介導跨上皮轉運,觀察其對變應性鼻炎的影響。 材料和方法：6-8周齡的雌性BALB/c小鼠,用卵清蛋白腹腔注射致敏,經(jīng)鼻滴入激發(fā)建立小鼠變應性鼻炎模型。應用免疫組化和Western Blot檢測CD23在小鼠鼻腔黏膜的表達。經(jīng)鼻滴入CD23抗體B3B4阻斷CD23-IgE介導跨上皮轉運,同時應用同種型抗體和經(jīng)鼻類固醇激素作為治療的陰性和陽性對照。經(jīng)鼻干預后,我們通過評估變應性鼻炎癥狀和病理組織學改變觀察干預效果；同時通過觀察血清和鼻腔灌洗液OVA特異性IgE、LTC4、ECP和IL-4水平,鼻黏膜組織中ECP水平和脾細胞反應情況評估干預效果。 結果：CD23結構性的表達于小鼠鼻黏膜上皮細胞,在過敏狀態(tài)下其表達發(fā)生了上調；在體內(nèi),CD23在鼻腔黏膜上皮細胞的上調與小鼠血清IL-4水平呈正相關,小鼠鼻腔灌洗液中IgE水平與CD23在鼻腔黏膜上皮細胞表達的量亦呈正相關。經(jīng)鼻吸入CD23抗體B3B4后,小鼠鼻部癥狀減輕；組織學檢查發(fā)現(xiàn),鼻腔黏膜下嗜酸性粒細胞浸潤減少；ELISA檢測結果發(fā)現(xiàn)血清及鼻腔灌洗液OVA特異性IgE、LTC4、ECP和IL-4水平降低；Western Blot檢測發(fā)現(xiàn)鼻黏膜組織ECP水平同樣降低；流式細胞術示B3B4經(jīng)鼻吸入干預糾正了CD4+輔助性T細胞反應向Th2細胞表型的偏移。這些結果提示在體內(nèi)經(jīng)鼻吸入CD23抗體干預抑制變應性鼻炎小鼠的炎癥反應。 結論：我們的研究提示CD23介導的跨上皮轉運IgE和IgE變應原免疫復合物在變應性鼻炎發(fā)病機制中起重要作用,鼻黏膜上皮表達的CD23可作為變應性鼻炎治療的新靶點。
[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.

【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R765.21

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本文編號：1691549