摘要
前言
支气管哮喘是以气道可逆性阻塞、气道高反应性和淋巴细胞、嗜酸性粒细胞浸润气道粘膜下层为特征性的慢性气道炎症。关于支气管哮喘气道炎症的发病机制,研究普遍认为主要是免疫源性炎症。多数学者认为CD4+辅助性T淋巴细胞两种亚型(Th1和Th2)的失衡是哮喘发生的重要机制,具体表现为Th2细胞的活化亢进,这是哮喘发病机制中的关键环节。
近年来,CD4+辅助性T淋巴细胞的另一种亚型Th17细胞在支气管哮喘发病机制中的作用日益受到重视。Th17区分于Thl和Th2的特点在于其特异性高水平分泌IL-17,是一种新型的促炎CD4+T效应细胞。转化生长因子-p和IL-6是诱导Th17细胞分化的关键因子。目前研究认为Th17细胞产生分泌的IL-17可以通过诱导释放的促炎症因子和促中性粒细胞动员的细胞因子协调局部组织的炎症,在哮喘过敏性气道炎症中发挥重要作用。
p-抑制蛋白属于抑制蛋白家族,包括p-抑制蛋白1和p-抑制蛋白2。p-抑制蛋白是众所周知的G蛋白偶联受体的负性调节蛋白,但同时作为支架蛋白和连接蛋白,参与受体在胞内的信号转导。有研究认为p-抑制蛋白2参与过敏性哮喘的发病机制,而且是在炎症反应的上游调节过敏性哮喘的发病。
p-抑制蛋白在参与受体在胞内的信号转导时,他们募集内吞蛋白和信号分子与受体结合,从而活化促分裂原活化蛋白激酶(MAPK)信号通路。MAPK信号通路包括ERK1/2, p38和JNK等几条通路,其中ERK1/2是MAPK级联反应中研究最为活跃,也是最为重要的信号传导通路之一。有研究证实在小鼠淋巴结细胞中IL-17的产生和表达可通过促分裂原活化蛋白激酶(MAPK)信号通路调控。
p-抑制蛋白2参与过敏性哮喘的发病机制,且是在炎症反应的上游调节过敏性哮喘的发病,那它是否通过调节IL-17的表达和产生而发挥效用呢?β-抑制蛋白可以活化促分裂原活化蛋白激酶(MAPK)信号通路,而在小鼠淋巴结细胞中IL-17的产生和表达可通过促分裂原活化蛋白激酶(MAPK)信号通路调控,且有研究发现β-抑制蛋白2能部分通过活化ERK1/2信号通路刺激IL-6的产生,而IL-6是诱导Thl7细胞分化的关键因子,所以我们假设p-抑制蛋白2可能通过刺激CD4+T细胞表达和产生IL-17参与过敏性哮喘的发病,且其对IL-17的作用可能部分是通过活化ERK1/2信号通路来实现的。
由此,本实验分三个部分：(1)建立急性哮喘小鼠动物模型,并证明急性哮喘小鼠模型建立成功；(2)接下来研究急性哮喘小鼠脾源性CD4+T细胞中p-抑制蛋白2表达的变化和p-抑制蛋白2对哮喘小鼠脾源性CD4+T细胞表达和产生IL-17的影响；(3)最后研究p-抑制蛋白2是否通过活化ERK1/2蛋白促进急性哮喘小鼠脾源性CD4+T细胞表达和产生IL-17,从而探讨p-抑制蛋白2促进急性哮喘小鼠脾源性CD4+T细胞表达和产生IL-17的可能机制。本实验希望通过研究p-抑制蛋白2对哮喘小鼠CD4+T细胞表达和产生IL-17的影响及其可能机制为哮喘防治提供新思路和理论依据。
第一章急性哮喘小鼠模型的建立与评价
目的建立急性哮喘小鼠动物模型,完善急性哮喘小鼠动物模型的评价标准,并鉴定急性哮喘小鼠模型建立是否成功。
方法将20只SPF级BABL/c小鼠随机分为两组：急性哮喘组(15只)以OVA致敏、激发；正常对照组(5只)以等体积的PBS代替OVA致敏、激发；两组均在末次激发24h后处理小鼠。处理包括：1.查看小鼠的一般行为活动；2.乙酰甲胆碱激发小鼠后,有创肺阻抗法测定小鼠的气道反应性；3.BALF行细胞学分类、计数；4.观察肺组织的病理变化；5.计算脾源性CD4+T细胞总数。
结果
(1)急性哮喘组小鼠出现类似于人的哮喘发作症状,而正常对照组小鼠表现基本正常。
(2)与正常对照组相比,急性哮喘组小鼠肺阻力(RL)对乙酰甲胆碱的浓度反应曲线明显上移(P<0.01)；急性哮喘组小鼠肺动态顺应性(Cdyn)对乙酰甲胆碱的浓度反应曲线明显下移(P<0.01)。
(3)与正常对照组相比,急性哮喘组小鼠肺组织支气管及血管周围大量炎性细胞浸润(P<0.01),气道上皮可见较多黏液分泌。
(4)急性哮喘组小鼠BALF白细胞总数及巨噬细胞、中性粒细胞、嗜酸性粒细胞和淋巴细胞的计数明显高于正常对照组(P均<0.01)。
(5)急性哮喘组小鼠脾源性CD4+T细胞数明显高于正常对照组(P<0.01)。
结论
(1)建立了急性哮喘小鼠动物模型,并且模型鉴定成功；
(2)脾源性CD4+T细胞数可以做为急性哮喘小鼠动物模型的评价标准之一。
第二章β-抑制蛋白2对急性哮喘小鼠脾源性CD4+T细胞表达和产生IL—17的影响
目的探讨急性哮喘小鼠脾源性CD4+T细胞中β-抑制蛋白2和IL-17表达的变化以及β-抑制蛋白2对急性哮喘小鼠脾源性CD4+T细胞表达和产生IL-17的影响。
方法首先研磨小鼠脾脏制成单细胞悬液后裂解红细胞,再予免疫磁珠分离小鼠脾源性CD4+T细胞,悬于加有胎牛血清的RPMI1640培养液中。正常对照组和哮喘组小鼠脾源性CD4+T细胞分别加入ConA(?)口PMA刺激培养24小时后行real-time PCR(?)(?)western blot测定细胞中β-抑制蛋白2的表达。再通过基因瞬时转染的方法,将三对siRNA-β-抑制蛋白2片段和阴性对照片段分别转染入急性哮喘小鼠脾源性CD4+T细胞后,RT-PCR检测β-抑制蛋白2mRNA的表达,筛选出其中具有最佳沉默效果的片段。Western blot检测阴性对照组、具有最佳沉默效果片段组哮喘小鼠脾源性CD4+T细胞中β-arrestin2的蛋白的表达。其后小鼠脾源性CD4+T细胞分为正常对照组、哮喘组和转染组(转染最佳沉默片段哮喘组),分别加入ConA(?)(?)PMA刺激培养24小时后行real-time PCR(?)(?)western blot测定细胞中IL-17mRNA和蛋白的表达,ELISA法检测细胞培养上清中IL-17的浓度。
结果
(1)哮喘组小鼠脾源性CD4+T细胞β-抑制蛋白2mRNA和蛋白的表达高于正常对照组(P均<0.01)；
(2)在三对siRNA片段中以siRNA-β-抑制蛋白2-1123沉默效果最为明显(P<0.01)；
(3)哮喘组小鼠脾源性CD4+T细胞中IL-17mRNA和蛋白的表达高于正常对照组(P均<0.01)；哮喘组小鼠脾源性CD4+T细胞培养上清中IL-17的浓度高于正常对照组(P<0.01)；
(4)转染组哮喘小鼠脾源性CD4+T细胞中IL-17mRNA和蛋白的表达低于哮喘组(P<0.01)；转染组哮喘小鼠脾源性CD4+T细胞培养上清中IL-17的浓度低于哮喘组(P<0.01)
结论
(1)急性哮喘小鼠脾源性CD4+T细胞中β-抑制蛋白2的表达增高；
(2)急性哮喘小鼠脾源性CD4+T细胞中IL-17的表达和产生增高；
(3)β-抑制蛋白2促进急性哮喘小鼠脾源性CD4+T细胞IL-17的表达和产生。
第三章p-抑制蛋白2促进急性哮喘小鼠脾源性CD4+T细胞表达和产生IL—17的机制研究
目的：探讨p-抑制蛋白2是否通过活化ERK1/2蛋白刺激急性哮喘小鼠脾源性CD4+T细胞表达和产生IL-17。
方法：本部分实验分两小部分。①为探讨急性哮喘小鼠脾源性CD4+T细胞中p-抑制蛋白2对活化ERK1/2蛋白表达的影响,同第二部分,取正常对照组、哮喘组和转染组小鼠脾源性CD4+T细胞,分别加入ConA和PMA刺激培养24小时后行western blot测定细胞中活化ERK1/2蛋白的表达。②为探讨急性哮喘小鼠脾源性CD4+T细胞中活化ERK1/2蛋白对表达和产生IL-17的影响,取小鼠脾源性CD4+T细胞分三个组：正常对照组、哮喘组和PD98059组(ERK1/2抑制剂PD98059预处理急性哮喘小鼠脾源性CD4+T细胞2小时),ConA和PMA刺激培养24小时后,行real-time PCR和western blot分别检测三组细胞中IL-17mRNA口蛋白的表达,ELISA法检测培养上清中IL-17的浓度。
结果：
(1)哮喘组小鼠脾源性CD4+T细胞活化ERK1/2蛋白的表达高于正常对照组(P<0.01)；
(2)转染组小鼠的脾源性CD4+T细胞中活化ERK1/2蛋白的表达低于哮喘组(P<0.01)；
(3)PD98059组哮喘小鼠脾源性CD4+T细胞中IL-17mRNA和蛋白的表达低于哮喘组(P均<0.05)；PD98059组哮喘小鼠脾源性CD4+T细胞培养上清中IL-17的浓度低于哮喘组(P<0.05)。
结论：
(1)急性哮喘小鼠脾源性CD4+T细胞中活化ERK1/2的表达增高；
(2)活化ERK1/2促进急性哮喘小鼠脾源性CD4+T细胞IL-17的表达和产生；
(3)p-抑制蛋白2促进急性哮喘小鼠脾源性CD4+T细胞ERK1/2的活化,活化ERK1/2通路是p-抑制蛋白2促进急性哮喘小鼠脾源性CD4+T细胞表达和产生IL-17的机制之-
Preface
Bronchial asthma is chronic airway inflammation with the characteristics of reversible airway obstruction, bronchial hyperresponsiveness and airway submucosal infiltration of lymphocytes and eosinophil. Regarding the induction of airway inflammation, it was widely recognized that it is a result of immune response in the bronchial airway. Many experts believe that imbalance of two types of CD4+T-helper cells, Thl and Th2, is the underlying cause for asthma. Specifically, the increased activity of Th2cells is a key event in asthma induction.
In recent years, the roles of another type of CD4+T-helper cells, Th17, in airway inflammation are receiving increasing attention. Th17is characterized by the secretion of Interleukin-17(IL-17). It has been recognized as a novel pro-inflammatory CD4+T effector cell. TGF-β and IL-6are essential for Th17cell differentiation. IL-17is a cytokine that discovered recently, belongs to a unique type of multi-functional cytokines, which coordinates the local inflammation of tissues by inducing the release of proinflammatory factors and mobilizing neutrophils, so it plays an important role in allergic airway inflammation in asthma.
β-arrestins, members of the arrestin family of proteins, are designated consist of (3-arrestin1and β-arrestin2. They are initially known as negative regulators of GPCR-mediated signaling. We now know that they also serve as scaffolds and adapters in receptor endocytosis and signal transduction. One study had shown that β-arrestin2was essential to the development of allergic asthma and it exerted its regulatory effect at a proximal step in the inflammatory cascade.
β-arrestins recruit endocytic proteins and a variety of signaling molecules to the receptors, thus activate mitogen-activated protein kinase (MAPK) cascades including extracellular signal-regulated kinase1/2 (ERK1/2), Jun N-terminal kinase (JNK), one of the p38kinases, and Src family kinases. ERK1/2is the one of the most important signaling pathways. One study had shown that IL-17production and expression in murine lymph node cells can regulated by utilization of MAPK (ERK1/2, p38and JNK) signaling.
Since that β-arrestin2was essential to the development of allergic asthma and it exerted its regulatory effect at a proximal step in the inflammatory cascade, whether β-arrestin2take effect by regulating IL-17production and expression? Recently one study has demonstrated that β-arrestin2positively mediated IL-6production which is very important for the IL-17production and this was mediated, in part, by the ERK1/2signaling pathway.We hade known that (3-arrestins activated mitogen-activated protein kinase (MAPK) cascades and IL-17production and expression in murine lymph node cells can regulated by utilization of MAPK (ERK1/2, p38and JNK) signaling, So,we propose a hypothesis that β-arrestin2may regulate IL-17expression and production partly through ERK1/2signaling pathway in allergic asthma.
To confirm this hypothesis, our research was divided into three parts:(1) establish acute mouse model of asthma and confirm reliability of the model with modified evaluation criterion.(2) investigate the change of β-arrestin2expression and IL-17expression in acute mouse model of asthma and elucidate the effect of β-arrestin2on IL-17expression and production in spleen CD4+T lymphocytes from acute mouse model of asthma.(3) investigate whether (3-arrestin2stimulated IL-17expression and production of spleen CD4+T lymphocytes in a murine asthma model mediated by ERK1/2activation to confirm underlying mechanisms of β-arrestin2stimulating Interleukin-17expression and production of Spleen CD4+T lymphocytes in a murine asthma model. Through this study, we hope to provide further experimental evidence for therapeutic treatment of asthma.
Part I Establish acute mouse model of asthma and confirm reliability of the model
Objective:To establish acute mouse model of asthma and confirm reliability of the model with modified evaluation criterion.
Methods:20SPF level female BABL/c mice were randomly divided into normal control group and asthma group, with15mice in asthma group and5mice in normal control group. The asthma model was established by sensitization with intraperitoneal injection of ovalbumin (OVA) and aerosol challenge with repeated inhalation of OVA in the asthma group. The control group received PBS as the substitution of OVA. After24hours of the last inhalation, asthmatic symptoms were observed; The changes in airway response were determined by lung resistance(RL) and Lung dynamic compliance (Cdyn) stimulated by Methacholine(Mch); Lung tissue sections were staininged for general pathology; The white cell count, cell counts of neutrophils, eosinophils, lymphocytes and macrophages of bronchoalveolar lavage fluid (BALF) were measured; The cell counts of spleen CD4+T lymphocytes was detected.
Results:
(1) Asthma symptoms were more severe in asthma group compared with normal group.
(2) The dose-response curves for RL was obviously shifted upward in asthma group compared with normal group(p<0.01);while the dose-response curves for Cdyn was significantly shifted downward in asthma group (p<0.01).
(3) Total white cell counts, cell counts of neutrophils, eosinophils, lymphocytes and macrophages of bronchoalveolar lavage fluid (BALF) significantly increased in asthmatic mice than those of normal control group (respectively p<0.01).
(4) There were more extensive inflammatory cells infiltration around the bronchi, and mucus excretion in airway lumen was found in asthma group compared with normal control group(p<0.01).
(5) The cell counts of spleen CD4+T lymphocytes in asthma group were obviously higher than those in normal control group(p<0.01).
Conclusions:
(1)The reliability of the established acute mouse model of asthma was successfully confirmed.
(2) The cell counts of spleen CD4+T lymphocytes can become one of evaluation criterions of acute asthmatic mouse model.
Part Ⅱ The effect of β-arrestin2on interleukin-17expression and production in spleen CD4+T lymphocytes from acute mouse model of asthma
Objective:To investigate the change of β-arrestin2expression and IL-17expression and production in acute mouse model of asthma, and to elucidate the effect of β-arrestin2on IL-17expression and production in spleen CD4+T lymphocytes from acute mouse model of asthma.
Methods:
Grinding spleens of mice from asthmatic group and normal group into spleen mononuclear cells suspension. After removing red blood cells from the suspension, CD4+T lymphocytes were isolated from spleen mononuclear cells with immuno-magnetic beads, and then were cultivated in RPMI-1640with10%Fetal Bovine Serum and stimulated by ConA and PMA. After24hours, the expression of β-arrestin2protein in CD4+T lymphocytes was detected by Western blot, the expression of β-arrestin2mRNA in CD4+T lymphocytes was detected by Realtime PCR. Next, Splenic CD4+T lymphocytes from acute mouse model of asthma were transfected with three chemosynthesis β-arrestin2siRNA sequences and negative control sequences. The best efficient siRNA-β-arrestin2was chosed by detecting the β-arrestin2mRNA expression by RT-PCR. β-arrestin2protein of the best efficient transfected group and the negative control group were measured by Western blot. Then the best siRNA-β-arrestin2was transfected into splenic CD4+T lymphocytes from asthma group, the CD4+T lymphocytes from normal group、asthma group and transfected group were stimulated by ConA and PMA for24hours. the expression of IL-17protein in CD4+T lymphocytes was detected by Western blot, the expression of IL-17mRNA in CD4+T lymphocytes was detected by Realtime PCR, the accumulation of IL-17in supernatants from cultures of CD4+T lymphocytes were measured by ELISA.
Results:
(1) The expression of β-arrestin2mRNA and protein in spleen CD4+ T lymphocytes from asthma group was increased compared with those from normal control group(respectively p<0.01);
(2) siRNA-β-arrestin2-1123had the best silencing effect among all siRNA sequences(p<0.01);
(3) The expression of IL-17mRNA and protein in spleen CD4+T lymphocytes from asthma group was increased compared with those from normal control group(respectively p<0.01); The accumulation of IL-17in supernatants from cultures of CD4+T lymphocytes from asthma group was increased compared with those from normal control group(p<0.01);
(4) The expression of IL-17mRNA and protein in spleen CD4+T lymphocytes from transfected group was lower in comparison with those from asthma group(respectively p<0.01); The accumulation of IL-17in supernatants from cultures of CD4+T lymphocytes from transfected group was lower in comparison with those from asthma group(p<0.01).
Conclusions:
(1) β-arrestin2expression was increased in spleen CD4+T lymphocytes from acute mouse model of asthma.
(2) IL-17expression and production was increased in spleen CD4+T lymphocytes from acute mouse model of asthma.
(3) β-arrestin2increased IL-17expression and production of spleen CD4+T lymphocytes in a murine asthma model.
Part Ⅲ The underlying mechanism of β-arrestin2stimulating interleukin-17expression and production of spleen CD4+T lymphocytes in a murine asthma model
Objective:To investigate whether β-arrestin2stimulated IL-17expression and production of spleen CD4+T lymphocytes in a murine asthma model mediated by ERK1/2activation.
Methods:(1) Firstly to elucidate the effect of β-arrestin2on activated ERK1/2protein(phosphorylated-ERK1/2) expression in spleen CD4+T lymphocytes from acute mouse model of asthma, CD4+T lymphocytes from normal group、asthma group and transfected group were stimulated by ConA and PMA for24hours. Then, phosphorylated-ERK1/2(p-ERK1/2) protein expression of CD4+T lymphocytes was detected by Western blot.(2) Next to elucidate the effect of activated ERK1/2on IL-17expression and production in spleen CD4+T lymphocytes from acute mouse model of asthma, CD4+T lymphocytes from acute mouse model of asthma were pretreated with the ERK1/2inhibitor, PD98059. CD4+T lymphocytes from normal group, asthma group and PD98059group were stimulated by ConA and PMA for24hours. Then, the expression of IL-17protein in CD4+T lymphocytes was detected by Western blot, the expression of IL-17mRNA in CD4+T lymphocytes was detected by Realtime PCR, the accumulation of IL-17in supernatants from cultures of CD4+T lymphocytes were measured by ELISA.
Results:
(1) The expression of activated ERK1/2protein in spleen CD4+T lymphocytes from asthma group was increased compared with those from normal control group(p<0.01);
(2) The expression of activated ERK1/2protein in spleen CD4+T lymphocytes from transfected group was lower in comparison with those from asthma group(p<0.01);
(3) The expression of IL-17mRNA and protein in spleen CD4+T lymphocytes from PD98059group was lower in comparison with those from asthma group(respectively p<0.05); The accumulation of IL-17in superatants from cultures of CD4+T lymphocytes from PD98059group was lower in comparison with those from asthma group(p<0.05).
Conclusions:
(1) Activated ERK1/2expression was increased in spleen CD4+T lymphocytes from acute mouse model of asthma;
(2) Activated ERK1/2increased IL-17expression and production of spleen CD4+T lymphocytes in a murine asthma model;
(3) β-arrestin2increased activated ERK1/2expression of spleen CD4+T lymphocytes in a murine asthma model; Activating ERK1/2expression is one of the mechanisms of β-arrestin2increasing IL-17expression and production of CD4+T lymphocytes in a murine asthma model.
引文
[1]. Fireman P. Understanding asthma pathophysiology. Allergy Asthma Proc,2003, 24(2):79-83.
[2]. Hahn C, Islamian AP, Renz H, et al. Airway epithelial cells produce neurotrophins and promote the survival of eosinophils during allergic airway inflammation.J Allergy Clin Immunol,2006,117(4):787-794.
[3]. Ngoc PL, Gold DR, Tzianabos AO, et al. Cytokines, allergy and asthma. Curr Opin Allergy Clin Immunol,2005,5(2):161-166.
[4]. Careau E, Proulx LI, Pouliot P, et al.Antigen sensitization modulates alveolar macrophage functions in an asthma model. Am J Physiol Lung Cell Mol Physiol,2006,290(5):L871-9.
[5]. Duan W, Aguinaldo Datiles AM, Leung BP, et al.An anti-inflammatory role for a phosphoinositide 3-kinase inhibitor LY294002 in a mouse asthma model.Int Immunopharmacol,2005,5(3):495-502.
[6]. Chiba Y, Ueno A, Sakai H, et al. Hyperresponsiveness of bronchial but not tracheal smooth muscle in a murine model of allergic bronchial asthma. Inflamm Res,2004,53(11):636-642.
[7]. Choi JH, Oh SW, Kang MS, et al.Trichostatin A attenuates airway inflammation in mouse asthma model.Clin Exp Allergy,2005,35(1):89-96.
[8]. Tong J, Bandulwala HS, Clay BS, et al. Fas-positive T cells regulate the resolution of airway inflammation in a murine model of asthma.J Exp Med, 2006,203(5):1173-1184.
[9]. Schramm CM, Guernsey L, Secor E, et al. Tolerance induced by chronic inhaled antigen in a murine asthma model is not mediated by endotoxin.Biochim Biophys Acta,2006,1762(5):499-501.
[10]. Yang G, Li L, Volk A, et al. Therapeutic dosing with anti-interleukin-13 monoclonal antibody inhibits asthma progression in mice.J Pharmacol Exp Ther,2005,313(1):8-15.
[11]. Temelkovski J, Hogan SP, Shepherd DP, et al.An improved murine model of asthma:selective airway inflammation, epithelial lesions and increased methacholine responsiveness following chronic exposure to aerosolised allergen. Thorax,1998,53(10):849-856.
[12]. Harrington LE, Hatton RD, Mangan PR, et al. Interleukin 17-producingCD4+ effector T cells develop via a lineage distinct from the T helper typel and 2 lineages. Nat Immunol,2005,6(11):1123-1132.
[13]. North ML, Khanna N, Marsden PA, et al. Functionally Important Role for Arginase 1 in the Airways Hyperresponsiveness of Asthma. Am J Physiol Lung Cell Mol Physiol, Mar 2009,296(6):911-920.
[14]. Locke NR, Royce SG, Wainewright JS, et al. Comparison of Airway Remodeling in Acute, Subacute, and Chronic Models of Allergic Airways Disease. Am J Respir Cell Mol Biol,2007,36:625-632.
[15]. Wild JS, Sigounas A, Sur N, et al. IFN- gamma -inducing Factor(IL-18) increases allergic sensitization, serum IgE, Th2 cytokines, and airway eosinophilia in a mouse model of allergic asthma.J Immunol,2000 Mar 1, 164(5):2701-2710.
[16]. Guo M, Huang T, CuY i, et al. PrPC interacts with tetraspanin-7 through bovine PrP154-182 containing alpha-helix 1. Biochem Biophys Res Commun, Jan 2008,365(1):154-157.
[17]. Ferrada MA, Gordon EL, Jen KY, et al. (R)-albuterol decreases immune responses:role of activated T cells. Respir Res, Jan 2008,9:3.
[18].陈黎,吕祖铭.哮喘动物模型制作的进展.泸州医学院学报,2002年,25(2)：173-175.
[19]. Kumar RK, Foster PS. Modeling allergic asthma in mice:pitfalls and opportunities.Am J Respir Cell Mol Biol,2002,27(3):267-272.
[20]. Kips JC, Anderson GP, Fredberg JJ, et al. Murine models of asthma.Eur Respir J,2003,22(2):374-382.
[21].沈华浩,王苹莉.支气管哮喘小鼠模型应用评价.中华结核和呼吸杂志,2005,28(4)：284-286.
[22]. Fu CL, Ye YL, Lee YL, et al. Effects of overexpression of IL-10, IL-12,TGF-beta and IL-4 on allergen induced change in bronchial responsiveness.Respir Res,2006,7:72.
[23]. Fang SP, Tanaka T, Tago F, et al. Immunomodulatory effects of gyokuheifusan on INF-gamma/IL-4(Th1/Th2) balance in ovalbumin (OVA)-induced asthma model mice. Biol Pharm Bull,2005,28(5):829-833.
[24]. Bakir M, Tukenmez F, Bahceciler NN, et al. Heat-killed Mycobacteriumbovis-bacillus Calmette Guerin-suppressed total serum IgE response in ovalbumin sensitized newborn miee. J Asthma, 2000,37(4):329-334.
[25]. Wills-Karp M, Ewart SL.The genetics of allergen-induced airway hyperresponsiveness in mice.Am J Respir Crit Care Med,1997,156(4 Pt 2):S89-96.
[26]. Tanaka H. Functional molecules in allergic bronchial asthma.Yakugaku Zasshi,2002,122(9):637-642.
[27]. Oliveira FA, Lima-Jumior RC, Cordeiro WM, et al. Pentacyclic triterpenoids, alpha, beta-amurins, suppress the scratching behavior in a mouse model of prurutus. Pharmacol Biochem Behav,2004,78(4):719-725.
[28].施焕中.正确认识和合理应用支气管哮喘的动物模型.中华结核和呼吸杂志,2005,28(11)：749-750.
[29].何权瀛.对我国哮喘动物实验研究的评价与思考.国外医学呼吸系统分册,2005,25(9)：718-719.
[30]. Alder A, Cieslewicz G, Irvin CG. Unrestrained plethysmography is an unreliable measure of airway responsiveness in BALB/c and C57BL/6 mice. J Appl Physiol,2004,97(1):286-292.
[31]. Brown RH, Walters DM, Greenberg RS, et al. A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice. J Appl Physiol,1999,87(6):2362-2365.
[32]. Murphy K M, Reiner S L. The lineage decisions of helper T cells.Nat Rev Immunol,2002,2:933-944
[33]. Mosmann T R, Coffman R L. Thl and Th2 cells:Different patterns of lymphokine secretion leads to different functional properties. Annu Rev Immunol,1989,7:145-73
[34]. Sakaguchi S. Regulatory T cells:Key controllers of immunologic self-tolerance. Cell,2000,101:455-458
[35]. Park H, Li Z, Yang X O, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat Immunol,2005,6(11): 1133-1141
[36]. Kay AB. The role of T lymphocytes in asthma. Chem Immunol Allergy,2006, 91:59-75.
[37]. Herrick CA, Bottomly K. To respond or not to respond:T cells in allergic asthma. Nat Rev Immunol,2003,3 (5):405-412.
[38]. Gor DO, Rose NR, Greenspan NS. Th12Th2:a Procrustean paradigm. Nat Immunol,2003,4 (6):503-505.
[39]. Harrington LE, Hatton RD, Mangan PR, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat Immunol,2005,6(11):1123-32.
[40]. Nakae S, Komiyama Y, Nambu A, et al. Antigen-specific T cell sensitization is impaired in IL-17-deficient mice, causing suppression of allergic cellular and humoral responses. Immunity,2002,17(3):375-87.
[41]. Hellings PW, Kasran A, Liu Z, et al. Interleukin-17 orchestrates the granulocyte influx into airways after allergen inhalation in a mouse model of allergic asthma. Am J Respir Cell Mol Biol,2003,28(1):42-50.
[42]. Oda N, Canelos PB, Essayan DM, et al. Interleukin-17F induces pulmonary neutrophilia and amplifies antigen-induced allergic response. Am J Respir Crit Care Med,2005,171(1):12-8.
[43]. Phyllis F. Y. Cheung, Chun K. Wong, and Christopher W. K. Lam. Molecular Mechanisms of Cytokine and Chemokine Release from Eosinophils Activated by IL-17A, IL-17F, and IL-23:Implication for Th17 Lymphocytes-Mediated Allergic Inflammation. Immunol,Apr 2008,180:5625-5635.
[44]. Molet S, Hamid Q, Davoine F, et al. IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines. J Allergy Clin Immunol,2001,108(3):430-8.
[45]. Zhao Y, Yang J, Gao YD, et al.Th17 immunity in patients with allergic asthma. Int Arch Allergy Immunol,2010,151(4):297-307.
[46]. Louis R, Lau LC, Bron AO, et al. The relationship between airways inflammation and asthma severity. Am J Respir Crit Care Med,2000,161(1): 9-16.
[47]. Kingsmore SF, Peppel K, Suh D, et al. Genetic mapping of the beta-arrestin 1 and 2 genes on mouse chromosomes 7 and 11 respectively. Mamm Genome,1995,6(4):306-307.
[48]. Claing A., Laporte SA, Caron MG, et al. Endocytosis of G protein-coupled receptors:roles of G protein-coupled receptor kinases and betaarrestin proteins. Prog Neurobiol,2002,66,61-79.
[49]. Reiter E, Lefkowitz RJ. GRKs and beta-arrestins:roles in receptor silencing, trafficking and signaling. Trends Endocrinol Metab,2006,17(4):159-65.
[50]. Lefkowitz RJ, Whalen EJ. beta-arrestins:traffic cops of cell signaling. Curr Opin Cell Biol,2004,16(2):162-8.
[51]..Lefkowitz RJ, Shenoy SK. Transduction of receptor signals by beta-arrestins. Science,2005,308(5721):512-7.
[52]. Avissar S, Matuzany-Ruban A, Tzukert K, et al. Beta-arrestin-1 levels:reduced in leukocytes of patients with depression and elevated by antidepressants in rat brain. Am J Psychiatry,2004,161(11):2066-2072.
[53]. Tsutsui S, Vergote D, Shariat N, et al. Glucocorticoids regulate innate immunity in a model of multiple sclerosis:reciprocal interactions between the A1 adenosine receptor and beta-arrestin-1 in monocytoid cells. FASEB J, 2008,22(3):786-796.
[54]. Luan B, Zhao J, Wu H, et al. Deficiency of a beta-arrestin-2 signal complex contributes to insulin resistance. Nature,2009,457(7233):1146-1149.
[55]. Kim J, Zhang L, Peppel K, et al. Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration. Circ Res,2008,103(1):70-79.
[56]. Porter KJ, Gonipeta B, Parvataneni S, et al. Regulation of lipopolysaccharide-induced inflammatory response and endotoxemia by beta-arrestins. J Cell Physiol,2010,225(2):406-416.
[57]. Jeong HC, Lee SY, Lee EJ, et al. Proteomic analysis of peripheral T-lymphocytes in patients with asthma. Chest,2007,132(2):489-496.
[58]. Walker JK, Fong AM, Lawson BL, et al. Beta-arrestin-2 regulates the development of allergic asthma. J Clin Invest,2003,112(4):566-574.
[59]. Factor P. Gene therapy for asthma. Mol. Therapy,2003,7(2):148-152.
[60]. Oboki K, Ohno T, Saito H, et al. Th17 and allergy. Allergol Int,2008,57:121-134.
[61]. Varga EM, Nouri-Aria KT, Till SJ, et al. Immunomodulatory treatmentstrategies for allergic diseases. Curr Drug Targets Inflamm Allergy,2003,2:31-46.
[62]. Barczyk A, Pierzchala W, Sozanska E. Interleukin-17 in sputum correlates with airway hyperresponsiveness to methacholine. Respir Med,2003 97:726-733.
[63]. Chakir J, Shannon J, Molet S, et al. Airway remodeling associatedmediators in moderate to severe asthma:effect of steroids on TGF-β, IL-11,IL-17, and type Ⅰ and type Ⅲ collagen expression. J Allergy Clin Immunol,2003, 111:1293-1298.
[64]. Bullens DM, Truyen E, Coteur L, et al. IL-17 mRNA in sputum of asthmatic patients:linking T cell driven inflammation and granulocytic influx. Respir Res,2006,7:135.
[65]. Sun YC, Zhou QT, Yao WZ. Sputum interleukin-17 is increased and associated with airway neutrophilia in patients with severe asthma. Chin Med J (Engl),2005,118:953-956.
[66]. Langrish C L, Chen Y, Blumenschein W M, et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. J Exp Med,2005,201: 233-240
[67]. Veldhoen M, Hocking R J, Atkins C J, et al. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity,2006,24:179-189
[68]. Bettelli E, Carrier Y, Gao W, Korn T, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature,2006,441(7090):235-8.
[69]..Mangan PR, Harrington LE, O'Quinn DB, et al. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature,2006, 441(7090):231-4.
[70]. Wan S, Xia C, Morel L. IL-6 produced by dendritic cells from lupus-prone mice inhibits CD4+CD25+ T cell regulatory functions. J Immunol,2007, 178(1):271-279
[71]. Stojanovic I, Cvjeticanin T, Lazaroski S, et al. Macrophage migration inhibitory factor stimulates interleukin-17 expression and production in lymph node cells. Immunology.2009,126(1):74-83.
[72]. Bogoyevitch MA, Court NW. Counting on mitogen-activated protein kinases-ERKs 3,4,5,6,7 and 8.Cell signal,2004,16(12):1345-1354.
[73]. Roux PP, Blenis J. ERK and p38MAPK-activated protein kinases:a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev,2004,68(2):320-344.
[74]. Lefkowitz RJ, Shenoy SK. Transduction of receptor signals by beta-arrestins. Science,2005,308(5721):512-517.
[75]. Wei H, Ahn S, Shenoy SK, et al. Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin Ⅱ activation of extracellular signal-regulated kinases 1 and 2. Proc Natl Acad Sci U S A,2003,100(19): 10782-10787.
[76]. Ahn S, Shenoy SK, Wei H, et al. Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin Ⅱ receptor. J Biol Chem,2004,279(34):35518-35525.
[77]. Shenoy SK, Drake MT, Nelson CD, et al. beta-arrestin-dependent, G protein-independent ERK 1/2 activation by the beta2 adrenergic receptor. J Biol Chem,2006,281(2):1261-1273.
[78]. McDonald PH, Chow CW, Miller WE, et al. Beta-arrestin 2:a receptor-regulated MAPK scaffold for the activation of JNK3. Science, 2000,290:1574-1577.
[79]. Sun Y, Cheng Z, Ma L, et al. Beta-arrestin2 is critically involved in CXCR4-mediated chemotaxis, and this is mediated by its enhancement of p38 MAPK activation. J Biol Chem,2002,277:49212-49219.
[80]. Fan H, Luttrell LM, Tempel GE, et al. Beta-arrestins 1 and 2 differentially regulate LPS-induced signaling and pro-inflammatory gene expression. Mol Immunol,2007,44(12):3092-3099.
[81]. Jang BC, Lim KJ, Paik JH, et al. Up-regulation of human beta-defensin 2 by interleukin-lbeta in A549 cells:involvement of PI3K, PKC, p38 MAPK, JNK, and NF-kappaB. Biochem Biophys Res Commun,2004,320(3):1026-33.
[82]. Luttrell LM, Roudabush FL, Choy EW, et al. Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds. Proc Natl Acad Sci U S A,2001,98(5):2449-54.
[83]..DeFea KA, Vaughn ZD, O'Bryan EM, et al. The proliferative and antiapoptotic effects of substance P are facilitated by formation of a beta-arrestin-dependent scaffolding complex. Proc Natl Acad Sci U S A.2000,97(20):11086-91.
[84]..DeFea KA, Zalevsky J, Thoma MS, et al. beta-arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol,2000,148(6):1267-81.
[1]. Kingsmore SF, Peppel K, Suh D, et al. Genetic mapping of the beta-arrestin 1 and 2 genes on mouse chromosomes 7 and 11 respectively. Mamm Genome, 1995,6(4):306-307.
[2]. Granzin J, Wilden U, Choe HW, et al. X-ray crystal structure of arrestin from bovine rod outer segments. Nature,1998,391(6670):918-921.
[3]. Sutton RB, Vishnivetskiy SA, Robert J, et al. Crystal structure of cone arrestin at 2.3A:evolution of receptor specificity. J Mol Biol,2005,354(5):1069-1080.
[4]. Wang P, Wu Y, Ge X, et al. Subcellular localization of beta-arrestins is determined by their intact N domain and the nuclear export signal at the C terminus. J Biol Chem,2003,278(13):11648-11653.
[5]. Nobles KN, Guan Z, Xiao K, et al. The active conformation of beta-arrestin1: direct evidence for the phosphate sensor in the N-domain and conformational differences in the active states of beta-arrestins1 and-2. J Biol Chem, 2007,282(29):21370-21381.
[6]. Milano SK, Kim YM, Stefano FP, et al. Nonvisual arrestin oligomerization and cellular localization are regulated by inositol hexakisphosphate binding. J Biol Chem,2006,281(14):9812-9823.
[7]. Hanson SM, Van Eps N, Francis DJ, et al. Structure and function of the visual arrestin oligomer. EMBO J,2007,26(6):1726-1736.
[8]. Imamoto Y, Tamura C, Kamikubo H, et al. Concentration-dependent tetramerization of bovine visual arrestin. Biophys J,2003,85(2):1186-1195.
[9]. Perry SJ, Baillie GS, Kohout TA, et al. Targeting of cyclic AMP degradation to beta 2-adrenergic receptors by beta-arrestins. Science,2002,298(5594):834-836.
[10]. Nelson CD, Perry SJ, Regier DS, et al. Targeting of diacylglycerol degradation to M1 muscarinic receptors by beta-arrestins. Science,2007,315(5812):663-666.
[11]. Ferguson SS, Downey WE,3rd, Colapietro AM, et al. Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization. Science, 1996,271(5247):363-366.
[12]. Laporte SA, Oakley RH, Zhang J, et al. The beta2-adrenergic receptor /betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc Natl Acad Sci U S A,1999,96(7):3712-3717.
[13]. Lin FT, Krueger KM, Kendall HE, et al. Clathrin-mediated endocytosis of the beta-adrenergic receptor is regulated by phosphorylation/dephosphorylation of beta-arrestin1. J Biol Chem,1997,272(49):31051-31057.
[14]. Girnita L, Shenoy SK, Sehat B, et al.{beta}-Arrestin is crucial for ubiquitination and down-regulation of the insulin-like growth factor-1 receptor by acting as adaptor for the MDM2 E3 ligase. J Biol Chem,2005,280(26):24412-24419.
[15]. Luttrell LM, Ferguson SS, Daaka Y, et al. Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science, 1999,283(5402):655-661.
[16]. Wei H, Ahn S, Shenoy SK, et al. Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin Ⅱ activation of extracellular signal-regulated kinases 1 and 2. Proc Natl Acad Sci U S A,2003,100(19):10782-10787.
[17]. Ahn S, Shenoy SK, Wei H, et al. Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin Ⅱ receptor. J Biol Chem,2004,279(34):35518-35525.
[18]. Shenoy SK, Drake MT, Nelson CD, et al. beta-arrestin-dependent, G protein-independent ERK1/2 activation by the beta2 adrenergic receptor. J Biol Chem, 2006,281(2):1261-1273.
[19]. Gesty-Palmer D, Chen M, Reiter E, et al. Distinct beta-arrestin-and G protein-dependent pathways for parathyroid hormone receptor-stimulated ERK1/2 activation. J Biol Chem,2006,281(16):10856-10864.
[20].Ebisuya M, Kondoh K, Nishida E. The duration, magnitude and compartmentalization of ERK MAP kinase activity:mechanisms for providing signaling specificity. J Cell Sci,2005,118(Pt 14):2997-3002.
[21]. Shenoy SK, McDonald PH, Kohout TA, et al. Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin. Science, 2001,294(5545):1307-1313.
[22]. Wang P, Gao H, Ni Y, et al. Beta-arrestin 2 functions as a G-protein-coupled receptor-activated regulator of oncoprotein Mdm2. J Biol Chem, 2003,278(8):6363-6370.
[23]. Gao H, Sun Y, Wu Y, et al. Identification of beta-arrestin2 as a G protein-coupled receptor-stimulated regulator of NF-kappaB pathways. Mol Cell, 2004,14(3):303-317.
[24]. Witherow DS, Garrison TR, Miller WE, et al. beta-Arrestin inhibits NF-kappaB activity by means of its interaction with the NF-kappaB inhibitor IkappaBalpha. Proc Natl Acad Sci U S A,2004,101 (23):8603-8607.
[25]. Wang Y, Tang Y, Teng L, et al. Association of beta-arrestin and TRAF6 negatively regulates Toll-like receptor-interleukin 1 receptor signaling. Nat Immunol,2006,7(2):139-147.
[26]. Kang J, Shi Y, Xiang B, et al. A nuclear function of beta-arrestin1 in GPCR signaling:regulation of histone acetylation and gene transcription. Cell, 2005,123(5):833-847.
[27]. Neuhaus EM, Mashukova A, Barbour J, et al. Novel function of beta-arrestin2 in the nucleus of mature spermatozoa. J Cell Sci,2006,119(Pt 15):3047-3056.
[28]. Avissar S, Matuzany-Ruban A, Tzukert K, et al. Beta-arrestin-1 levels:reduced in leukocytes of patients with depression and elevated by antidepressants in rat brain. Am J Psychiatry,2004,161(11):2066-2072.
[29]. Tsutsui S, Vergote D, Shariat N, et al. Glucocorticoids regulate innate immunity in a model of multiple sclerosis:reciprocal interactions between the A1 adenosine receptor and beta-arrestin-1 in monocytoid cells. FASEB J, 2008,22(3):786-796.
[30].Jeong HC, Lee SY, Lee EJ, et al. Proteomic analysis of peripheral T-lymphocytes in patients with asthma. Chest,2007,132(2):489-496.
[31]. Walker JK, Fong AM, Lawson BL, et al. Beta-arrestin-2 regulates the development of allergic asthma. J Clin Invest,2003,112(4):566-574.
[32].Luan B, Zhao J, Wu H, et al. Deficiency of a beta-arrestin-2 signal complex contributes to insulin resistance. Nature,2009,457(7233):1146-1149.
[33]. Kim J, Zhang L, Peppel K, et al. Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration. Circ Res,2008,103(1):70-79.
[34], Porter KJ, Gonipeta B, Parvataneni S, et al. Regulation of lipopolysaccharide-induced inflammatory response and endotoxemia by beta-arrestins. J Cell Physiol,2010,225(2):406-416.