【作者】 刘阳；

【导师】 崔永华；

【作者基本信息】 华中科技大学 ， 耳鼻咽喉—头颈外科， 2010， 博士

【摘要】 [研究背景及目的]变应性鼻炎(Allergic Rhinitis, AR)在发展中国家和欧美地区的流行情况非常严重,我国亦是变应性鼻炎高发地区,人群患病率约10%。临床上缺乏特异而有效的治疗靶点和干预手段。变应性鼻炎是由于机体在外界环境的诱导下导致的以鼻黏膜局部以TH2反应为主要特征的病理改变。在其病理过程中有大量的细胞因子和细胞的参与。基因芯片结果显示Clara细胞10KDa蛋白(CC10蛋白)为变应性鼻炎患者鼻黏膜中下调最明显的蛋白。CC10蛋白为新近认识的子宫珠蛋白超家族主要成员之一。它主要表达于与外界相通的上皮,包括支气管上皮和鼻粘膜上皮等。CCl0蛋白具有抗炎和免疫调节作用。既往研究显示它可以拮抗分泌性磷脂酸A2的活性,降低炎性细胞趋化,下调TH2细胞的分化,并且可以阻断前列腺素D2受体介导的NF-κB活性。我们以前的研究显示CC10基因敲除小鼠在抗原刺激下,也是产生过度的以TH2细胞反应为主的嗜酸性粒细胞炎症,并且CC10蛋白可以直接抑制TH2细胞因子的产生。辅助性T细胞17(TH17)细胞是最近几年发现的一种新型的辅助性T细胞。TH17细胞的发现源于对实验性自身免疫性脑脊髓炎(EAE),以及对胶诱导的关节炎(CIA)的研究。传统上认为,上述两种自身免疫病是由TH1细胞介导的。然而研究发现：清除或中和TH1型细胞因子IFN-γ或IL-12的功能,并不能预防或减轻疾病的进程。而清除IL-23的功能则延缓了疾病的进程。进一步发现,缺失IL-17+T细胞可以使EAE等自身免疫病的发病受到抑制,认识到是IL-17+T细胞而不是经典的TH1细胞在此环境中诱导自身免疫病。TH17细胞分泌的细胞因子除了IL-17(IL-17A)外,还包括IL-17F,以及IL-21、IL-22、IL-6、TNF-α等细胞因子。不同的T细胞亚群也有相对特异性的标记物,主要是它们的转录调控因子的不同。如TH1细胞为T-bet, TH2细胞为GATA-3,Tregs为FoxP3,目前证实TH17细胞的特异性转录调控因子为ROR-γt。TH17细胞在发现早期就已被证实与自身免疫病有着非常密切的关系。在各种自身免疫病,包括系统性红斑狼疮(SLE)以及肿瘤中都证实TH17能促进疾病的发生和发展。新近研究发现哮喘患者以及变应性鼻炎患者外周血中IL-17的含量是增高的,并且证实了在哮喘小鼠模型中TH17细胞是促进TH2细胞引起的嗜酸性粒细胞炎症。鉴于CC10蛋白对辅助性T细胞的作用以及TH17细胞在变应性呼吸道疾病中的作用,我们建立变应性鼻炎小鼠模型,探讨变应性鼻炎模型中CC10蛋白对TH17细胞的作用,为变应性疾病治疗开发CC10蛋白药物提供理论基础。具体研究目标如下：1.建立变应性性鼻炎模型①在疾病进程中动态观察鼻黏膜局部CC10蛋白与TH17细胞反应的关系。②研究CC10基因敲除变应性鼻炎小鼠模型中鼻黏膜局部TH17细胞反应的情况。③研究体内给予CC10蛋白对TH17细胞反应的影响。④研究CC10蛋白对TH17细胞反应的细胞机制和分子机制。[研究方法]1.采用卵清白蛋白皮下注射致敏并通过鼻腔进行激发建立野生型和CC10基因敲除变应性鼻炎小鼠模型,在疾病进程中对小鼠鼻黏膜组织行HE染色和PAS染色观察其病理改变,免疫组化方法检测鼻黏膜局部CC10蛋白表达情况。并通过Real-time PCR和ELISA方法检测TH1,TH2以及TH17细胞反应的相关的细胞因子水平。流式细胞术检测鼻黏膜局部TH17和TH2细胞反应情况。2.在CC10基因敲除变应性鼻炎小鼠模型致敏阶段腹腔给予CC10蛋白,小鼠鼻黏膜组织行HE染色和PAS染色观察其病理改变,并通过Real-time PCR和ELISA方法检测TH1,TH2以及TH17细胞反应的相关的细胞因子水平。流式细胞术检测腹部沟淋巴结以及鼻黏膜局部TH17和TH2细胞反应情况。3.在野生型变应性鼻炎小鼠模型激发阶段鼻腔给予CC10蛋白,小鼠鼻黏膜组织行HE染色和PAS染色观察其病理改变,并通过Real-time PCR和ELISA方法检测TH1,TH2以及TH17细胞反应的相关的细胞因子水平。流式细胞术检测鼻黏膜局部TH17和TH2细胞反应情况。4.分离野生型小鼠脾脏树突状细胞,体外给予CC10蛋白和卵清白蛋白进行刺激,ELISA方法检测培养的树突状细胞上清中IL-6, TGF-β以及IL-23的量。5.体外给予CC10蛋白作用后的树突状细胞和卵清白蛋白体内致敏后的T细胞进行作用,胞内因子染色检测TH17细胞的情况。6荧光双染检测CC10基因敲除小鼠和野生型小鼠变应性鼻炎模型小鼠脾脏中树突状细胞分泌IL-6, TGF-β以及IL-23的情况。7免疫组化方法检测CC10基因敲除小鼠和野生型小鼠变应性鼻炎模型中鼻黏膜局部上皮表达CCL20情况。体外培养BEAS-2B细胞,给予TH1,TH2,TH17细胞因子以及前炎细胞因子进行刺激,然后给予CC10蛋白进行处理,检测CC10蛋白对细胞因子引起的上皮细胞表达CCL20的作用。[实验结果]1.在野生型变应性鼻炎小鼠模型中,鼻粘膜上皮中CC10阳性细胞数目随着激发次数的增加,呈现逐渐下降的趋势。在激发前,鼻粘膜上皮中的CC10阳性细胞数目为100±2个／mm,在激发1天后,鼻粘膜上皮中的CC10阳性细胞数目为90±3个／mm,而在激发第3天和第5天以后,鼻粘膜上皮中的CC10阳性细胞数目分别为80±6个／mm和50±4个／mm,与生理盐水组相比,有显著统计学差别(p<0.05)。2.在野生型变应性鼻炎小鼠模型中,鼻粘膜局部TH17细胞相关分子如RORγt,IL-17A,IL-17F和IL-22mRNA表达水平分别比生理盐水对照组增加了3倍(p<0.05),4倍(p<0.01),5倍(p<0.05)和7倍(p<0.05)。而小鼠鼻腔灌洗液中的IL-17A的蛋白水平随着激发次数的增加,也呈现逐渐增加的趋势,其在第14天为200±2pg/ml和第15天为200±10pg/ml,与对照组相比,没有统计学差别；而在第17天为400±7pg/ml,第19天为1600±11pg/ml,与对照组相比,有明显统计学差别,分别为p<0.05和p<0.01。而在鼻腔灌洗液中,分泌IL-17的细胞主要为TH17细胞(CD4+IL-17+),所占比例为11.1%,与生理盐水组有明显统计学差别(p<0.05)。3.在CC10基因敲除变应性鼻炎小鼠模型中,鼻腔灌洗液中的细胞计数显示lml灌洗液中其总炎性细胞个数为1.75×106个,嗜酸性粒细胞数(0.9×106个／ml)和淋巴细胞计数(0.7×106个／ml),与对照组相比,有明显统计学差别。鼻粘膜局部IL-4,IL-5,IL-13的mRNA水平相对于野生型对照组有明显统计学差别(p<0.05),而IFN-Y的mRNA表达没有统计学差别。而在鼻腔灌洗液中,TH17细胞(CD4+IL-17+),所占比例为27.2%,与野生型对照组相比有明显统计学差别(p<0.05)。4.CCl0基因敲除变应性鼻炎小鼠模型中,分别在第0天和第7天致敏小鼠,于第14天处死小鼠,分离腹股沟淋巴结并分离其单个核细胞,流式细胞术显示TH2细胞比例为20.3%,TH17细胞比例为17.9%,与野生型小鼠相比,有明显统计学差别(p<0.05),而TH1细胞在两组之间没有统计学差别。5.在致敏阶段给予CC10基因敲除小鼠CC10蛋白,抗原激发后鼻粘膜上皮中的杯状细胞数目,鼻腔灌洗液中的总炎性细胞,嗜酸性粒细胞和淋巴细胞数目相对于生理盐水对照组,都有显著性统计学差别。鼻粘膜局部IL-4,IL-5,IL-13的mRNA水平相对于野生型对照组有明显统计学差别(p<0.05),而IFN-γ的mRNA水平没有统计学差别。在鼻腔灌洗液中,TH17细胞(CD4+IL-17+)所占比例为20.2%,与生理盐水对照组(5.3%)相比有明显统计学差别。6.磁珠分选野生型未致敏小鼠脾脏中的T淋巴细胞,体外给予IL-4的中和抗体,IFN-Y的中和抗体,TGF-β,IL-6,IL-23细胞因子以及抗CD3单克隆抗体和抗CD28单克隆抗体使T细胞向TH17细胞分化,然后加入CC10蛋白,结果显示IL-4中和抗体,IFN-γ的中和抗体,TGF-β,IL-6和IL-23以及抗CD3单克隆抗体和抗CD28单克隆抗体联合使用明显使T细胞向TH17细胞分化,但是CC10蛋白不能抑制此作用。7.分别在第0天和第7天致敏小鼠,于第14天处死小鼠,分离小鼠脾脏组织,其组织切片行免疫双染显示在CC10基因敲除小鼠中树突状细胞分泌的TGF-β, IL-6和IL-23的水平显著高于野生型小鼠。8.磁珠分选野生型致敏小鼠的脾脏中的CD11C+树突状细胞,体外给予CC10蛋白和卵清白蛋白进行刺激,24小时后用ELISA方法检测上清中的TGF-β和IL-6的水平,与为给予CC10蛋白相比,加入CC10蛋白后,TGF-β和IL-6的水平是显著降低的,并且树突状细胞中的IL-23p19的mRNA水平也显著降低。9.卵清白蛋白致敏后的野生型小鼠中,将体外脾脏分离的树突状细胞用CC10蛋白处理后与卵清白蛋白致敏后的野生型小鼠脾脏分离T淋巴细胞进行混合培养,培养48小时后,流式细胞仪技术胞内细胞因子方法检测TH17细胞的比例,发现CC10蛋白处理后其比例为7.6%,而不予处理时为20.1%。10.在激发阶段给予野生型小鼠CC10蛋白,抗原激发后鼻粘膜上皮中的杯状细胞数目,鼻腔灌洗液中的总炎性细胞,嗜酸性粒细胞和淋巴细胞数目相对于生理盐水对照组,都有显著性统计学差别。鼻粘膜局部IL-4,IL-5,IL-13的mRNA水平相对于野生型对照组有明显统计学差别(p<0.05),而IFN-γ的mRNA表达没有统计学差别。在鼻腔灌洗液中,TH17细胞(CD4+IL-17+)所占比例为12%,与生理盐水对照组(5.3%)相比有明显统计学差别。11.鼻腔灌洗液的细胞滴片中,免疫荧光双染显示CC10基因敲除小鼠中CD11C阳性树突状细胞分泌的IL-23显著高于野生型对照组,并且Real-time PCR结果显示鼻粘膜局部IL-23p19和IL-23P40的mRNA水平显著高于野生型对照组。12.CC10基因敲除变应性鼻炎小鼠模型中,免疫组化显示鼻粘膜局部CCL20蛋白阳性表达细胞的数目显著高于对照组。体外CC10蛋白显著下调BEAS-2B细胞中由于各种细胞因子导致的CCL20的分泌。[结论]1.变应性鼻炎小鼠模型中鼻粘膜局部TH17细胞的数量增多以及其功能增强。2.CC10蛋白通过作用于树突状细胞,调节TH17细胞相关因子的表达来抑制致敏阶段TH17细胞的分化。3.CC10蛋白通过作用于树突状细胞调节其IL-23的分泌来抑制激发阶段TH17细胞的扩增。4.CC10蛋白是通过上皮细胞来调节TH17细胞的局部趋化。更多还原

【Abstract】 Background and Objective:Allergic rhinitits is very popular in developing countries and European countries and China as well. In China, the rate was 10%. In clinic, there are not proper therapeutic targets and preventive methods. TH2 related pathology was the cardinal feature of allergic rhinitis induced by environmental factors in the local epithelium and lot of cytokines and cell subsets contribute to the initiation and development of the disease. Gene microarry reveals that Clara cell 10-KDa Protein (CC10) was the most downregulated in the local mucosa of allergic rhinitis patients. CC10, a member of the secretoglobin family, also referred to as uteroglobin, is a steroid-inducible, multifunctional, secreted protein with anti-inflammatory and immunomodulatory effects. It is constitutively expressed by the epithelial lining of all organs that communicate with the external environment, including bronchi and nose. CC10 can inhibit the activity of phospholipase A2, suppress the expression and function of several cytokines, diminish inflammatory cell chemotaxis, downregulate Th2 cell differentiation, and block prostaglandin D2 receptor-mediated nuclear factor-κB activation. We and others previously also found that compared with responses seen in wild-type mice, CC10-deficient mice develop an intensive pulmonary inflammatory response after sensitization and challenge with Ag and CC10 protein could directly inhibits the differentiation of TH2 cell.TH17 cells are a new subset of T cells recongnized in recent years. TH17 cells are first discovered in experimental autoimmune encephalomyelitis (EAE), and arthritis (CIA). Traditionally, these two kinds of autoimmune disease are considered to be mediated by the TH1 cells. However, studies have found that:removal or neutralization TH1 related cytokines IFN-y or IL-12 could not prevent or mitigate the disease process. However, depeletion of IL-23 could slow the disease process. Subsequent studies confirmed that the lack of IL-23 reduced the in vivo IL-17+T percentage,but did not affect the number of Thl. Thus the correlation of IL-23 and IL-17+ cells in autoimmune disease are further confirmed and further studies found that lack of IL-17+T cells supressed the disease of EAE. Thus, it was th17 cells rather than classical TH1 cells in this environment induced autoimmune disease. In addition to the cytokines IL-17 (IL-17A), TH17 cells also secret IL-17F, and IL-21, IL-22, IL-6, TNF-a and other cytokines. Different T cell subsets have relatively specific markers in that they have different transcription factors. TH1 cells have T-bet, TH2 cells have GATA-3 and Tregs have FoxP3. It was confirmed that the specific transcription factor for Th17 cell was ROR-yt. In the early discovery TH17 cells have been tightly associated with autoimmune diseases including rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE) and TH17 have also been confirmed in the occurrence and development of tumors. Recent studies found that IL-17 levels are increased in patients with asthma and allergic rhinitis. Further investigation confirmed TH17 cells promote TH2 cell induced recruitment of eosinophils in a mouse model of asthma.In view of the role of CC10 protein in the differentiation of T helper cells and the possible role of TH17 cells in allergic airway diseases, we established a mouse model of allergic rhinitis for the study of the effect of CC10 on TH17 cells and provide a theoretical basis for developing CC10 protein drugs in clinic.Specific research objectives are as follows:1. To establish allergic rhinitis models①To observe the relationship between local CC10 protein and TH17 response in nasal mucosa.②To investigate the local Th17 response in local mucosa in CC10 knock out mouse models.③To study the effect of CC10 administration on Th17 response in vivo. ④To reveal the cell and molecular mechanisms.Methods:1. Subcutaneous injection of ovalbumin in sensitization and challenge with ovalbumin were exloited to establish wild-type and CC10 knockout mouse models of allergic rhinitis. The development of the disease process was discovered by HE staining or PAS staining. The expression of CC10 protein was detected by immunohistochemistry. Real-time PCR and ELISA methods were used to detect TH1, TH2, and TH17 response. flow cytometry method was used to detect the TH17 and TH2 response in nasal mucosa.2. CC10 was given intraperitoneally in the sensitized phase In the CC10 knockout mouse model of allergic rhinitis. The development of the disease process was discovered by HE staining or PAS staining. The expression of CC10 protein was detected by immunohistochemistry. Real-time PCR and ELISA methods were used to detect TH1, TH2, and TH17 response. flow cytometry method was used to detect the TH17 and TH2 response in nasal mucosa and inguinal lymph nodes.3. CC10 was given nasally in the challenge phase in wild type mouse model of allergic rhinitis. The development of the disease process was discovered by HE staining or PAS staining. The expression of CC10 protein was detected by immunohistochemistry. Real-time PCR and ELISA methods were used to detect TH1, TH2, and TH17 response. flow cytometry method was used to detect the TH17 and TH2 response in nasal mucosa and inguinal lymph nodes.4. wild-type mouse spleen dendritic cells were isolated and treated with CC10 protein and OVA in vitro, ELISA method was used to measure the level of IL-6, TGF-βin the supernatant and Real-time PCR method was used to measure the expression of IL-23 in dendritic cells.5. After in vitro treatment of dendritic cells with CC10 protein and ovalbumin, they were cocultured with T cells sensitized in vivo with OVA, Intracellular cytokine staining was used to detect Th17 response.6. Double Fluorescent staining was used to detect dendritic cells derived IL-6, TGF-βand IL-23 expression in the spleen in the allergic rhnitis models of CC10 gene knockout mice and wild-type mice. 7. Immunohistochemistry method was used to detect epithelial expression of CCL20 and Real-time PCR method was used to measure the expression of IL-23 in the nasal mucosa in CC10 gene knockout mice and wild-type mice model of allergic rhinitis. BEAS-2B cells in vitro were stimulated with TH1, TH2, and proinflammatory cytokines with the subsequent treatment with CC10.Results:1. In allergic rhinitis wild-type mice, the epithelial CC10 positive cells increased with the challenge times, in a gradual downward trend, before the challenge, the epithelial CC10-positive cell number was 100±2/mm, while one day after the first challenge, CC10-positive cells numbered 90±3/mm, and on day 17 and day 19, the CC10 positive cells were 80±6/mm and 50±4/mm. there was a significant statistical difference, compared with that of the saline group with P<0.05 and 0.01 respectively.2. In the wild-type allergic mouse model, local RORyt, IL-17A, IL-17F, IL-22 mRNA levels increased by3 fold (p<0.05),4 fold (p<0.01),5 fold (p<0.05) and 7 fold (p<0.05) respectively compared to the saline control group. IL-17A protein levels in the nasal lavage fluid increased with the challenge times, with a growing trend, On day 14 there was 200±2pg/ml and 200±10pg/ml on day 15, there was no statistical difference compared with the control group. there was statistically significant difference on 17 day (400±7pg/ml) and day19(1600±11pg/ml), compared with the control group with p <0.05 and p＜0.01 respectively. In the nasal lavage fluid, IL-17 was mainly produced by TH17 cells (CD4+IL-17+) which accounted for 10.2%, with statistically significant difference compared the saline group.3. In the CC10 knockout mouse model of allergic rhinitis, nasal lavage fluid cell counts showed that the total number of inflammatory cells were 1.75×106个,the number of eosinophils were 0.9×106, and lymphocytes were 0.7×106个, and there was statistically significant difference compared with the control group. there was statistically significant difference Nasal mucosa local IL-4, IL-5, IL-13 and mRNA levels (p<0.05) relative to wild-type control group and the expression of IFN-ymRNA has no significant difference. In the nasal lavage fluid, TH17 cells (CD4+IL-17+), accounted for 27.2%, and there was statistically significant difference compared with the wild-type control group. 4. After day 0 and 7 sensitization, mice were killed and inguinal lymph nodes were isolated. Mononuclear cells measured by flow cytometry method showed the proportion of TH2 cells was 20.3%, TH17 cells 17.9%, and there was statistically significant difference (p<0.05) compared to wild-type mice, while the percentage of TH1 cells had no significant difference between the two groups.5. When CC10 protein was administered in sensitized mice, after OVA challenge, the number of epithelial goblet cells, total inflammatory cells, eosinophils and lymphocytes in the nasal lavage were significantly decreased compared to those of saline control group, there was a significantly statistical difference. local IL-4, IL-5, IL-13 and mRNA levels of nasal mucosa was statistically significant different relative to wild-type control group (p <0.05) and the expression of IFN-γmRNA has no significant difference. In the nasal lavage fluid, TH17 cells (CD4+IL-17+)accounted for 20.2%, with the control group (5.3%) and there was statistically significant difference.6. Wild-type mouse spleen T lymphocytes were polarized with corresponding cytokine TGF-β, IL-6 and IL-23, IL-4 neutralizing antibody, IFN-y neutralizing antibody and anti-CD3 anti-CD28 monoclonal antibody to TH17 cells, and then the CC10 protein were treated. the result showed that TGF-β, IL-6 and IL-23, IL-4 neutralizing antibody, IFN-y neutralizing antibody and anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody significantly increase the differentiation of TH17 cells, but the CC10 protein could not inhibit this effect.7. At dayO and 7 after the two sensitization, the mice were killed and the mouse spleens were isolated. mouse spleen tissue sections by double staining showed that dendritic cells derived TGF-β, IL-6 and IL-23 levels in the CC10 knockout mice were significantly higher than thosein wild-type mice.8. Spleen CD11C+dendritic cells sorted by micobeads in wild-type sensitized mice were stimulated with CC10 protein and ovalbumin in vitro for 24 hours, TGF-β, IL-6 levels in the supernatant of the dendritic cells treated by CC10 protein were significantly reduced, and dendritic cell derived IL-23p19mRNA were also significantly lower.9. Dendritic cells treated with CC10 protein in vitro and OVA were cocultured with mouse spleen T cells from the sensitized mice for 48 hours, TH17 cells decreased from 20.1% to 7.6%. 10.When the mice were given CC10 protein in challenge stage, the number of goblet cells in the epithelium and total inflammatory cells, eosinophils and lymphocytes in nasal lavage significantly decreased compared to the number of saline control group. local IL-4, IL-5, IL-13 and mRNA levels in nasal mucosa was statistically significant differenr relative to that of wild-type control group (p<0.05) and the expression of IFN-ymRNA has no significant statistical difference. In the nasal lavage fluid, TH17 cells (CD4+IL-17+), accounted for 12%, with the control group (5.3%) and there was statistically significant difference.11. Nasal lavage cytoslides by immunofluorescence staining showed CD11C positive dendritic cells in CC10 knockout mice expressed significantly higher IL-23 than those in wild-type control group, Moreover, Real-time quantitative PCR method showed that local IL-23p19 and IL-23P40 mRNA level in the local mucosa was significantly higher than that of wild-type control group.12. Immunohistochemistry method showed that local CCL20 protein expression in nasal mucosa in CC10 gene knockout mouse model was significantly higher than that of the control. in vitro CC10 protein significantly downregulated BEAS-2B cell derived CCL20 induced by IL-β,TNF-α, IL-4, IL-13, IFN-γ.Conclusion:1. TH17 cells and the function were enhanced in the local mucosa in allergic rhinitis mouse model.2. CC10 protein inhibits TH17 cell differentiation by downregulating dendritic derived cytokines in the sensitization phase.3. CC10 protein inhibits the multiplication of Th17 cells in the challenge stage by regulating the secretion of IL-23 in dendritic cells.4. CC10 protein acts on the epithelial cells to regulate local TH17 cell chemotaxtics.更多还原