本文關(guān)鍵詞：白細(xì)胞介素-25在哮喘中通過(guò)誘導(dǎo)nuocytes促進(jìn)Th2型細(xì)胞因子表達(dá)的研究　出處：《山東大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： IL-25 nuocytes Th2型免疫應(yīng)答 IL-25抗體 哮喘

【摘要】：支氣管哮喘(asthma)是由多種炎癥細(xì)胞如嗜酸性粒細(xì)胞(Eosinophils,EOS).肥大細(xì)胞、T淋巴細(xì)胞等及多種細(xì)胞組分參與的氣道慢性炎癥性疾病,并且通常以氣道上皮的結(jié)構(gòu)改變?yōu)椴±硖卣�。支氣管哮喘的三個(gè)主要病理改變包括：氣道炎癥、氣道重塑及平滑肌功能紊亂。慢性炎癥反應(yīng)體現(xiàn)在細(xì)胞因子和細(xì)胞介質(zhì)的釋放,這些炎癥細(xì)胞因子和炎癥介質(zhì)可通過(guò)一定的途徑引起哮喘氣道炎性改變。哮喘使小氣道痙攣和黏液大量分泌,引起氣道狹窄和氣道高反應(yīng)。支氣管哮喘的病因及發(fā)病機(jī)制復(fù)雜,目前尚未完全明確,現(xiàn)今研究較多圍繞Th1/Th2免疫反應(yīng)失衡學(xué)說(shuō)。Th1/Th2平衡消長(zhǎng)是機(jī)體免疫反應(yīng)調(diào)節(jié)的基本方式,正常情況下,兩者處于一種平衡狀態(tài),Th1/Th2細(xì)胞之間處于相互抑制的狀態(tài),而遺傳變異及環(huán)境因素的變化可使兩者的平衡優(yōu)勢(shì)改變,從而導(dǎo)致Th1/Th2比例關(guān)系趨向于Th2占優(yōu)勢(shì),Th2相關(guān)的免疫反應(yīng)功能亢進(jìn)以及細(xì)胞因子分泌過(guò)多。CD4+Th2細(xì)胞活化釋放白細(xì)胞介素-5(Interleukin,IL.5)口白細(xì)胞介素-13(Interleukin,IL-13)等炎癥因子,促進(jìn)嗜酸性粒細(xì)胞向氣道募集、黏液過(guò)多分泌、上皮細(xì)胞增生肥大,促使B細(xì)胞轉(zhuǎn)換為IgE分泌型細(xì)胞,導(dǎo)致血清中IgE水平增加,引起氣道高反應(yīng)性。多項(xiàng)研究顯示,白細(xì)胞介素-25(Interleukin,IL.25)誘導(dǎo)Th2類型細(xì)胞因子如IL-5、IL-13產(chǎn)生,參與哮喘氣道炎癥的多個(gè)環(huán)節(jié),并參與支氣管哮喘引起的氣道重塑,與支氣管哮喘關(guān)系密切。研究還指出,固有免疫細(xì)胞也可生成IL-5和IL-13等Th2型細(xì)胞因子,因此推測(cè)在固有免疫細(xì)胞在Th2型免疫應(yīng)答的啟動(dòng)中可能發(fā)揮著重要作用.Nuocytes是目前發(fā)現(xiàn)的Ⅱ型固有淋巴細(xì)胞(Type II innate lymphoid cells,ILC2s)之一,最早在腸道中發(fā)現(xiàn),廣泛存在于骨髓、外周血、脾臟中,但數(shù)量較少。目前越來(lái)越多的研究證明固有免疫細(xì)胞在細(xì)胞免疫應(yīng)答中起重要作用。近年來(lái)發(fā)現(xiàn)nuocytes在細(xì)胞免疫應(yīng)答中具有促進(jìn)Th2型細(xì)胞因子表達(dá)的作用,在哮喘動(dòng)物實(shí)驗(yàn)中也發(fā)現(xiàn)nuocytes的存在,并發(fā)現(xiàn)該細(xì)胞表面IL-25受體高表達(dá),故nuocytes是否參與哮喘疾病中與Th2型細(xì)胞因子的形成及與IL-25的相互作用,成為哮喘疾病發(fā)生機(jī)制的研究熱點(diǎn),也為哮喘治療開(kāi)辟了新的方向。[研究目的]探討支氣管哮喘中IL-25及Th2型細(xì)胞因子表達(dá)的關(guān)系及其在哮喘氣道炎癥中的作用；并探討IL-25在支氣管哮喘中是否通過(guò)誘導(dǎo)nuocytes促進(jìn)Th2型細(xì)胞因子表達(dá)。[研究方法]將BALB/C小鼠隨機(jī)分為3組,分別命名為哮喘組、抗IL-25組和對(duì)照組,其中,給予哮喘組及抗IL-25組小鼠致敏后霧化吸入1%卵清蛋白(Ovalbumin, OVA)建造動(dòng)物哮喘模型實(shí)驗(yàn)組,對(duì)照組小鼠用同體積0.9%氯化鈉溶液霧化。另外,抗IL-25組經(jīng)鼻滴入抗IL-25抗體(0.5mg/只),哮喘組及對(duì)照組經(jīng)鼻注入等體積的0.9%氯化鈉溶液。根據(jù)小鼠臨床癥狀及肺組織病理變化等為標(biāo)準(zhǔn)判斷造模是否成功。取各組小鼠的肺泡灌洗液,用于分析各組小鼠肺泡灌洗液中的白細(xì)胞及嗜酸粒細(xì)胞計(jì)數(shù)及嗜酸粒細(xì)胞占白細(xì)胞總數(shù)的比例；解剖各組小鼠獲取肺組織標(biāo)本,切片固定后采用HE染色觀察各組小鼠肺組織病理學(xué)變化,應(yīng)用酶聯(lián)免疫反應(yīng)(ELISA)、Western blot及實(shí)時(shí)定量-PCR(RT-PCR)等實(shí)驗(yàn)方法與技術(shù)測(cè)定哮喘組、抗IL-25組和對(duì)照組各組肺泡灌洗液、肺組織中IL-25及IL-5、IL-13等Th2型細(xì)胞因子的表達(dá)情況；運(yùn)用流式細(xì)胞儀測(cè)定哮喘組、抗IL-25組和對(duì)照組三組小鼠肺泡灌洗液中nuocytes的數(shù)量。采用SPSS 17.0統(tǒng)計(jì)軟件包進(jìn)行統(tǒng)計(jì)學(xué)數(shù)據(jù)處理,所有數(shù)據(jù)均以均數(shù)和標(biāo)準(zhǔn)差標(biāo)示,計(jì)量資料用t檢驗(yàn),檢驗(yàn)水準(zhǔn)a=0.05,P0.05為有統(tǒng)計(jì)學(xué)意義。分析IL-25及Th2型細(xì)胞因子表達(dá)的關(guān)系及其在哮喘氣道炎癥中的作用；并探討IL-25在支氣管哮喘中是否通過(guò)誘導(dǎo)nuocytes促進(jìn)Th2型細(xì)胞因子表達(dá)。[結(jié)果](1)各組間肺泡灌洗液白細(xì)胞、嗜酸性粒細(xì)胞計(jì)數(shù)及嗜酸粒細(xì)胞比例：哮喘組小鼠肺泡灌洗液中白細(xì)胞總數(shù)、嗜酸性粒細(xì)胞絕對(duì)值及嗜酸性粒細(xì)胞百分比均較抗IL-25組和對(duì)照組小鼠明顯增高,差別有統(tǒng)計(jì)學(xué)意義(P0.05)；而抗IL-25組小鼠肺泡灌洗液中白細(xì)胞總數(shù)、嗜酸性細(xì)胞絕對(duì)值及嗜酸性粒細(xì)胞百分比與對(duì)照組小鼠相比無(wú)明顯差異(P0.05)。(2)HE染色哮喘組小鼠支氣管粘膜下和肺泡周圍募集大量炎癥細(xì)胞浸潤(rùn),黏液分泌細(xì)胞增多,支氣管中有大量分泌物聚集；抗IL-25組小鼠整個(gè)肺組織及支氣管周圍均有少量細(xì)胞核成藍(lán)染的炎性細(xì)胞聚集,少量的黏液分泌細(xì)胞增生,支氣管中有少量分泌物聚集,呈輕度炎性病變的表現(xiàn)；對(duì)照組小鼠肺組織標(biāo)本可見(jiàn)支氣管粘膜下和肺泡周圍無(wú)明顯的炎癥細(xì)胞浸潤(rùn)及分泌物。(3)通過(guò)酶聯(lián)免疫吸附試驗(yàn)(ELISA)發(fā)現(xiàn)：哮喘組小鼠肺泡灌洗液中IL-25、IL-5、IL-13的表達(dá)比抗IL-25組和對(duì)照組小鼠肺泡灌洗液中IL-25、IL-5、IL-13的表達(dá)明顯增高,有統(tǒng)計(jì)學(xué)意義(P0.05)；而抗IL-25組小鼠肺泡灌洗液中IL-25、IL-5、IL-13的表達(dá)比與對(duì)照組小鼠肺泡灌洗液中IL-25、IL-5、IL-13的表達(dá)相比無(wú)明顯差異(P0.05)。(4)通過(guò)WesternBlot及RT-PCR檢測(cè)發(fā)現(xiàn)：哮喘組小鼠肺組織中IL-25蛋白及mRNA的表達(dá)比抗IL-25組和對(duì)照組小鼠肺組織中IL-25蛋白及mRNA的表達(dá)明顯增強(qiáng),具有統(tǒng)計(jì)學(xué)意義(P0.05)；而抗IL-25組小鼠IL-25蛋白及mRNA的表達(dá)與對(duì)照組小鼠肺組織中IL-25蛋白及mRNA的表達(dá)水平相比,兩組間無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05)。(5)流式細(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)：哮喘組小鼠肺泡灌洗液中nuocytes的數(shù)量明顯增多,與抗IL-25組和對(duì)照組小鼠肺泡灌洗液中nuocytes的數(shù)量相比,有統(tǒng)計(jì)學(xué)意義(P0.05)；而抗IL-25組小鼠肺泡灌洗液中nuocytes的數(shù)量與對(duì)照組小鼠肺泡灌洗液中nuocytes的數(shù)量表達(dá)增多,但無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。[結(jié)論]支氣管哮喘模型中,支氣管粘膜下和肺泡周圍募集大量炎癥細(xì)胞浸潤(rùn),黏液分泌細(xì)胞增多。肺泡灌洗液中白細(xì)胞總數(shù)及嗜酸性粒細(xì)胞數(shù)量增加,支氣管中有大量分泌物聚集,哮喘小鼠肺泡灌洗液及肺組織中IL-25及Th2型炎性細(xì)胞因子IL-5、IL-13表達(dá)增多。哮喘小鼠肺泡灌洗液中固有免疫細(xì)胞nuocytes明顯增加,nuocytes表面可表達(dá)IL-25受體,應(yīng)用抗IL-25抗體干預(yù)后小鼠肺泡灌洗液中nuocytes數(shù)量及IL-5、IL-13等相關(guān)的炎癥指標(biāo)明顯減少,表明IL-25在哮喘中可通過(guò)誘導(dǎo)nuocytes促進(jìn)Th2型細(xì)胞因子表達(dá),抗IL-25抗體可阻斷這一途徑,有望成為哮喘治療的新靶點(diǎn)。
[Abstract]:Bronchial asthma (asthma) is a chronic inflammatory disease involving many inflammatory cells, such as Eosinophils, EOS, mast cells, T lymphocytes and many other cellular components. Three major pathological changes in bronchial asthma include airway inflammation, airway remodeling, and smooth muscle dysfunction. Chronic inflammatory reaction is reflected in the release of cytokines and cell mediators. These inflammatory cytokines and inflammatory mediators can lead to airway inflammatory changes through a certain way. Asthma makes small airway spasms and mucus secrete, causing airway stenosis and airway hyperresponsiveness. The etiology and pathogenesis of bronchial asthma is complex, and it is not completely clear at present. Nowadays, many studies have focused on the theory of Th1/Th2 immune response imbalance. Th1/Th2 balance is the basic method to regulate the body's immune response, under normal circumstances, both in a balanced state, the mutual inhibition between Th1/Th2 cells in the state, and changes in genetic variation and environmental factors to balance the advantages of both changed, which causes the Th1/ Th2 ratio tends to the dominance of Th2, Th2 related immune the reaction function hyperfunction and cytokine hypersecretion. CD4+Th2 cell activation and release of interleukin -5 (Interleukin, IL.5) white interleukin -13 (Interleukin, IL-13) and other inflammatory cytokines, promote eosinophil recruitment to the airway, mucus hypersecretion, epithelial hyperplasia, prompting B cells into IgE secreting cells, leading to increased levels of IgE in serum. Induced airway hyperresponsiveness. Many studies have shown that interleukin -25 (Interleukin, IL.25) induces Th2 type cytokines, such as IL-5 and IL-13, is involved in many links of airway inflammation in asthma, and is involved in airway remodeling induced by bronchial asthma, which is closely related to bronchial asthma. The study also pointed out that innate immune cells can also generate IL-5 IL-13 and Th2 type cytokines, presumably in innate immune cells may play an important role in.Nuocytes is found in type II cells in the innate immune response priming in type Th2 (Type II innate lymphoid cells, ILC2s) one of the first found in the intestinal tract that widely exist in bone marrow, peripheral blood and spleen, but fewer. At present, more and more studies have shown that innate immune cells play an important role in cellular immune response. In recent years, found that nuocytes could promote the expression of Th2 type cytokines on cell immune response, nuocytes also found in asthma animal experiments, and found that the high expression of the cell surface receptor IL-25, the formation of the involvement of nuocytes in asthma and Th2 type cytokines and their interaction with IL-25, become a hot research topic the pathogenesis of asthma, also opened up a new direction for the treatment of asthma. [Objective] to explore the relationship between the expression of IL-25 and Th2 cytokines in bronchial asthma and its role in airway inflammation in asthma, and to explore whether IL-25 can induce nuocytes expression in bronchial asthma by promoting the expression of Th2 cytokines. [Methods] BALB/C mice were randomly divided into 3 groups, which were named as asthma group, anti IL-25 group and control group, the asthma group and anti IL-25 group treated mice were sensitized after inhalation of 1% ovalbumin (Ovalbumin, OVA) the construction of the experimental animal model of asthma group, control group of mice with the same volume of 0.9% Sodium Chloride Solution atomization. In addition, the anti IL-25 group was injected with anti IL-25 antibody (0.5mg/ only) through nasal drip, and 0.9% Sodium Chloride Solution in asthma group and control group were injected into the nose by nasal injection. The success of the model was judged according to the clinical symptoms and pathological changes of the lung tissue. Take the mice alveolar lavage, analysis of bronchoalveolar lavage fluid in leukocyte and eosinophil count and eosinophil leukocyte accounted for the proportion of the total used; anatomy of mice lung tissue were obtained after fixation by HE staining, sections of lung tissue were observed pathological changes, application of ELISA the reaction (ELISA), Western blot and real-time -PCR (RT-PCR) and other experimental methods and techniques for determination of anti asthma group, IL-25 group and control group bronchoalveolar lavage and lung tissue in IL-25 lotion, and IL-5, IL-13 and Th2 type cytokines expression; using flow cytometry, anti asthma group IL-25 three groups of mice in the bronchoalveolar lavage fluid nuocytes. SPSS 17 statistical software package was used for statistical data processing. All data were marked by mean and standard deviation. T was used to test data, and a=0.05 was tested. P0.05 was statistically significant. Objective to analyze the relationship between IL-25 and Th2 cytokines expression and its role in airway inflammation in asthma, and to explore whether IL-25 can induce nuocytes expression in bronchial asthma. [results] (1) between the groups in bronchoalveolar lavage fluid of white blood cells, eosinophil count and eosinophil percentage of asthmatic mice in BALF leukocyte count and eosinophil absolute value and percentage of eosinophils were anti IL-25 group and control group were significantly higher, statistically the significance of difference (P0.05); anti IL-25 mice bronchoalveolar leukocytes and eosinophils in BALF and the absolute value of the percentage of eosinophils compared with control mice showed no significant difference (P0.05). (2) HE staining around the asthmatic mice bronchial mucosa and alveolar recruitment inflammatory cell infiltration, mucus secretion of cells increased, a large number of bronchial secretions aggregation; anti IL-25 mice of the bronchus and lung tissue surrounding the nucleus into the blue has a small amount of inflammatory cell aggregation, a small amount of mucus secreting cells in bronchial hyperplasia. A small amount of secretions aggregation, mild inflammatory lesions; the control group around the mouse lung tissue specimen showed bronchial mucosa and alveolar no obvious inflammatory cell infiltration and
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R562.25

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6 王烈;;小兒哮喘20年回顧[A];全國(guó)第26屆中醫(yī)兒科學(xué)術(shù)會(huì)暨王烈教授學(xué)術(shù)思想研討會(huì)論文集[C];2009年

7 孟憲武;孫志霞;;哮喘的誘因與證治[A];全國(guó)第26屆中醫(yī)兒科學(xué)術(shù)會(huì)暨王烈教授學(xué)術(shù)思想研討會(huì)論文集[C];2009年

8 王烈;;漫談哮喘家庭防治[A];全國(guó)第26屆中醫(yī)兒科學(xué)術(shù)會(huì)暨王烈教授學(xué)術(shù)思想研討會(huì)論文集[C];2009年

9 呂波;蔡定邦;許煊;梁星群;;100例小兒哮喘綜合治療管理研究[A];面向21世紀(jì)的科技進(jìn)步與社會(huì)經(jīng)濟(jì)發(fā)展（下冊(cè)）[C];1999年

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