【作者】 郭莹莹；

【导师】 王丽颖；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 支气管哮喘(简称哮喘)是一种常见的气道慢性炎症性疾病。T淋巴细胞及其分泌的多种细胞因子在哮喘的发病过程中起重要作用,表现为Th1/Th2细胞功能失衡且Th2型免疫反应占优势。大量哮喘动物实验研究表明,CpG ODN能够增强Th1型免疫反应进而抑制哮喘的特征性表现,包括减少气道嗜酸性粒细胞的炎性浸润、降低气道高反应性、下调Th2类细胞因子的表达以及降低血清中变应原特异性的IgE的水平。CpG ODN是一种含未甲基化CpG基序的寡脱氧核苷酸,通过模拟细菌的DNA片段经人工合成,具有广泛免疫调节作用。根据不同的结构特点和体外刺激作用,CpG ODN可分为A、B和C三种类型。Dynavax公司研制的ISS 1018是一种B型CpG ODN,临床前和临床试验已证实了ISS 1018对过敏性疾病有治疗作用。在本课题中,我们应用卵清白蛋白(OVA)诱导的急性哮喘小鼠模型,探讨了本科室自行设计的三种新型ODN(含CpG基序的B型CpG ODN和C型CpG ODN,不含CpG基序的抑制性ODN)对哮喘的治疗作用,并以ISS 1018为阳性对照。结果表明,不论是腹腔注射还是鼻腔滴入,C型CpG ODN均能显著减轻气道炎症反应、减少嗜酸性粒细胞的浸润和粘液分泌。在变应原致敏的同时给予C型CpG ODN,能够增强Th1型细胞因子的表达并促进IgG2a抗体的产生,还可能激活调节性T细胞发挥免疫抑制作用,进而下调了哮喘的Th2型优势免疫反应。经比较得出,C型CpG ODN对哮喘的治疗效果优于ISS 1018,提示本室拥有自主知识产权的C型CpG ODN可能成为一种治疗哮喘的新型有效的免疫调节剂。更多还原

【Abstract】 BackgroundBronchial asthma is a kind of chronic inflammatory airway disease which involves multiple cells and cellular components. The inflammatory cells include mast cells, eosinophils and T lymphocytes, and the cellular components consist of cytokines, chemokines, inflammatory mediators and so on. T lymphocytes and a variety of cytokines play an important role in the process of asthma. The immune changes of asthma is considered to be Th1/Th2 imbalance and Th2 immune response.dominatedCpG ODN, mimicing bacterial DNA fragments and containing unmethylated CpG motif, is a kind of synthetic oligodeoxynucleotide with immune stimulating effects. CpG ODN can be divided into three types including A, B and C. They are recognized by TLR9 expressed in endosomes of human B cells and plasmacytoid dendritic cells (pDC), and capable of stimulating NK cells, macrophages and cytotoxic T lymphocytes (CTL) indirectly. Thus CpG ODN could induce and enhance the innate immune response and acquired immune response, including Th1 biased immune response and T regulatory immune response. Animal experiments of asthma show that CpG ODN, either used alone or combined with allergen systemicly or locally, can prevent the occurrence of asthma by inhibiting the development of airway eosinophilic inflammatory infiltration, airway hyper-responsiveness (AHR) and the secretion of allergen-specific IgE and Th2-type cytokines. Therefore, CpG ODN displays promising prospect in treating asthma.PurposeIn this study, novel B- type CpG ODN, C-type CpG ODN and suppressive ODN designed by our lab were used in OVA-induced mouse model of acute asthma. By intraperitoneal injection, select a candidate CpG ODN with best therapeutic effect on asthma and preliminary explore the possible mechanism. In the following study, through nasal instillation, evaluate the effect of the candidate CpG ODN on treating asthma.Methods1 Establishment of asthma model and intervention by ODN.In the first part, thirty-six BALB/c mice were randomly divided into six groups, including OVA group, C-CpGgroup, B-CpG group, S-ODN group, ISS 1018 group and NS group,. On day 0, 7 and 14, by intraperitoneal injection, the mice were sensitized with OVA/Alum (10μg/1 mg, OVA group) or OVA/Alum/ODNs (10μg/1 mg/5μg, ODN groups), or NS/Alum (NS group) in 200μl of saline, respectively. From day 21, mice were challenged with 1%OVA solution in a self-made glass container for 20min each time during five consecutive days. Then mice were killed on day 26. In the second part, eighteen BALB/c mice were randomly divided into three groups, including OVA group, C-CpG group and NS group. On day 0, 7 and 14, mice in OVA group and C-CpG group were sensitized with OVA/Alum (10μg/1 mg) in 200μl of saline, and NS group with NS/Alum instead. On day 21, 22 and 23, mice in C-CpG group were treated by nasal instillation of C-CpG (15μg), NS and OVA group treated by NS instead. From day 25～28, mice were challenged using the previous method. Then mice were killed on day 29.2 Record of asthmatic symptoms in the process of OVA challenge.In the process of OVA atomization challenge, a variety of clinical symptoms of mice including irritability, shortness of breath, head and face itching, arched back, and nodding motion were observed during five consecutive days. And times of touching noses, times of spasm, latency of spasm and mean duration of each spasm were recorded under the blind principle.3 Detection of mRNA expression of cytokines by RT-PCR.Right lungs of mice were isolated, shredded and stored in Trizol reagent. Then transcription factors GATA-3 and T-bet, cytokines including IL-4、IFN-γ、IL-10 and TGF-βmRNA expression were detected by RT-PCR.4 Related detection of lung histopathology.The rest of lungs were fixed in 10% Formalin and prepared for pathlogical sections by HE staining and PAS staining. Pathological scores and eosinophils counts were analyzed in HE staining sections through the light microscope.5 Detection of OVA specific antibodies by indirect ELISA.Blood were collected by tail vein the day before each OVA sensitization and initial challenge, and by removing eyes on the final day. After sera were isolated, OVA specific IgG, IgG1 and IgG2a antibodies were detected by indirect ELISA.ResultsThe results of symptoms during the process of OVA challenge indicated that times of touching noses in model group had no significance compared with control group in two models. In the first model, times of spasm in model group become more, mean duration of each spasm become longer, and latency of spasm become shorter. While in the second model, times of spasm and latency of spasm in model group had no significance compared with control group. Using the quantitative indicators of symptoms to evaluate the success of asthma model should be questioned.Through histopathology of HE staining, the model group showed obvious bronchial epithelial damage, mucosal edema, bronchial mucus secretion and inflammatory cell infiltration peri-bronchial and peri-blood vessels. By PAS staining, the model group is strongly positive, with marked hyperplasia of goblet cells, accompanied by mucus hyper-secretion and mucus plug formation. In contrast, the control group showed no epithelial thickening, no airway inflammatory cell infiltration but structural integrity of the alveolar wall. And PAS staining is negative with no mucus secretion. Pathological scores and EOS counts in the model group were obviously higher than the control group. It is indicated that the established asthma model was a success and could be used to study the effect of CpG ODN on asthma.Compared with the model group, C-CpG and B-CpG group both significantly reduced airway EOS infiltrated inflammation and mucus secretion. The effects were better than ISS 1018 and S-ODN. C-CpG alone by nasal instillation also significantly reduced allergic airway inflammation in asthma.C-CpG and B-CpG induced lower levels of IL-4 and higher levels of T-bet、IFN-γ、IL-10 and TGF-β. C-CpG upregulated the ratio of T-bet/GATA-3 and IFN-γ/IL-4.At the time of OVA sensitization, intraperitoneal injection of all ODNs significantly increased the levels of OVA specific IgG. B-CpG、ISS 1018 and S-ODN increased IgG1. C-CpG and B-CpG increased IgG2a. C-CpG upregulated the ratio of IgG2a/IgG1. However, before OVA challenge, intranasal instillation of C-CpG had little effect on levels of IgG, IgG1 and IgG2a.ConclusionAt the time of OVA sensitization, intraperitoneal injection of C type CpG ODN, significantly reduces airway eosinophils infiltrated inflammation of asthma, and inhibit Th2-type cytokines. It increases Th1-type transcription factors and cytokines and induce high levels of OVA specific IgG2a. It could also stimulate T regulatory inhibited cytokines to regulate Th1/Th2 imbalanced immune response. C type CpG ODN show better effect than B type CpG ODN. Before OVA challenge, intranasal instillation of C type CpG ODN alone also reduces airway eosinophils infiltrated inflammation of asthma. Therefore, C type CpG ODN might be a novel immunomodulator to treat asthma.更多还原