【作者】 王凯；

【导师】 沈华浩；

【作者基本信息】 浙江大学 ， 内科学， 2007， 博士

【摘要】 研究背景过敏性哮喘是一种以气道慢性炎症、气道高反应性(AHR)、可逆性气道阻塞和粘液高分泌为特征的气道慢性疾病，病因复杂，发病机制尚不明确。近年来的研究显示，Eos是哮喘发病过程中主要的炎症细胞。过敏性哮喘发病时，抗原提呈细胞将抗原传递给T淋巴细胞并激活Th2细胞，后者释放炎症介质、细胞因子和趋化因子如白介素4(IL-4)、IL-5、Eotaxin等引起Eos在气道的募集和活化。Eos不仅可分泌血小板活化因子、白三烯等炎症因子，引起支气管平滑肌痉挛、气管微血管通透性增加、气道粘膜水肿等反应；激活的Eos还可释放嗜酸细胞阳离子蛋白、主碱基蛋白、嗜酸细胞神经毒素、嗜酸细胞过氧化酶等对上皮细胞产生直接而显著的细胞毒作用，使气道上皮细胞坏死脱落，基底层细胞暴露，神经末梢裸露，从而产生气道高反应性。在哮喘患者的血、痰、支气管肺泡灌洗液(BALF)中均可见Eos的升高。趋化因子是一类结构相似、分子量约为8—10KD，具有趋化功能的重要的细胞因子，通过作用于靶细胞表面的趋化因子受体发挥生物学效应。根据半胱胺酸的相邻位置关系分为多个亚族。其中C-C亚族在气道炎症性疾病中有着重要的作用。Eotaxin属于CC类趋化因子，通过唯一的受体CCR3特异的强有力的诱导嗜酸性粒细胞向特定组织迁移。其对嗜酸性粒细胞具有高度的选择性，在趋化嗜酸性粒细胞参与哮喘气道炎症过程中具有重要的作用。CCR3(CC chemokine rceptor3)即CC趋化因子受体3，为一种受体蛋白，主要介导细胞对包括Eotaxin、Eotaxin-2、Eotaxin-3、RANTES、MCP-3、MCP-4以及MIP-1等多种趋化因子的反应并产生细胞迁移、活化等效应，其主要表达在嗜酸性粒细胞、嗜碱性粒细胞以及肥大细胞、TH2细胞、巨噬细胞上。Eotaxin等炎症介质通过CCR3激活Eos，吸引大量Eos迁移到气道，导致气道炎症和气道高反应性的产生，引起哮喘的发作。因此，切断该炎症通路的信号传导将抑制气道炎症的发生。抗CCR3抗体可与Eos表面的CCR3受体特异性结合，阻断IL-5、Eotaxin等炎症因子对Eos的作用，使Eos无法向气道聚集，避免了Eos对气道的破坏和哮喘的发病。研究目的1．合成CCR3细胞外区NH2端多肽并以此多肽免疫小鼠，制备鼠抗人抗CCR3抗体。2．研究抗CCR3抗体对哮喘气道炎症和粘液高分泌的治疗作用。研究方法采用多肽合成仪经“固相合成法”合成了CCR3细胞外区NH2端30个氨基酸的多肽序列为mttsldtvet fgttsyyddv gllcekadtr，并进行多肽偶联和检测序列。以该多肽对BALB／c小鼠进行免疫，免疫以后眼球取血测定血清效价，血清里的抗体效价要在1∶6250以上取免疫小鼠脾细胞与骨髓瘤细胞SP2／0进行融合，筛选获得阳性克隆后产生并纯化抗体。选择健康雄性的C57BL／6小鼠，6—8周龄，体重18—22，分为4组。哮喘模型组(Ova组)：以卵白蛋白(Ova)作为抗原进行两次腹腔注射致敏(第0，14天)和三次雾化吸入激发(第24，25，26天)。抗CCR3抗体组(Anti—CCR3组)：Ova致敏和激发同Ova组，每次激发前腹腔注射抗CCR3抗体(PBND03，3mg／Kg)。非特异性IgG组(IgG组)：Ova致敏和激发同Ova组，每次激发前腹腔注射非特异性IgG(3mg／Kg)。空白对照组(Saline组)：以生理盐水代替Ova致敏和激发。末次激发48小时行肺泡灌洗，细胞计数，细胞因子检测，肺组织病理检查，MUC5AC基因表达检测。研究结果1．人CCR3 NH2端30个氨基酸多肽的合成采用多肽合成仪经固相合成法合成了人CCR3细胞外区NH2端30个氨基酸的多肽序列为mttsldtvet fgttsyyddv gllcedadtr，检测序列证实为目的序列。2．Western—Blot检测抗CCR3抗体特异性对杂交瘤细胞通过ELISA有限稀释法筛选阳性克隆，取阳性克隆里的上清，通过筛选共得到7个克隆。为了确定我们所得的抗体是抗CCR3的特异性抗体，我们采用了表面有丰富的CCR3的Daudi细胞破碎液对所制备的7个阳性克隆细胞产生的抗体进行Western—Blot分析。结果发现其中三个克隆的抗体PBND03、PBND04、PBND05在特定的大小的位置上有明显的显色条带，而其余4个克隆未见特异性条带。3．抗CCR3抗体的亲和力检测取PBND03、04、05细胞打腹水，得到腹水后取腹水，从中纯化单抗。对纯化好的抗体进行ELISA检测和浓度测定。结果为：PBND03亲和力＞1.28×10~9，PBND04亲和力≥2×10~7，PBND05亲和力≥3.2×10~8：ELISA最佳工作浓度分别为：0.1μ8、5μg、0.2μg：浓度分别为3.85mg／ml、2.63mg／ml、2.31mg／ml。由于PBND03的亲和力最高，该抗体进一步被用于在哮喘小鼠动物模型中研究对哮喘的治疗作用。4．抗CCR3抗体(PBND03)对BALF中炎症细胞的影响Ova组中的炎症细胞总数比Saline组中的明显增高。和Ova组相比，IgC组中的炎症细胞总数没有明显的变化，而Anti—CCR3组中的炎症细胞总数明显降低。分类计数结果显示，与Saline组相比较，Ova组中的Eos明显增高。Anti—CCR3组中的Eos较Ova组明显降低，而IgG组中的Eos与Ova组相比较却没有明显变化。各组间巨噬细胞、淋巴细胞均未见有显著性差异。5．抗CCR3抗体(PBND03)对Ova致敏和激发后导致的哮喘小鼠气道炎症的影响各组肺组织切片HE染色用来观察哮喘小鼠的气道炎症浸润情况以及抗CCR3抗体对气道炎症的抑制作用。Saline组：气道上皮完整，纤毛排列整齐，杯状细胞少见，基底层以及平滑肌层较薄，肺小血管内皮光滑，血管、气道周围见极少量的炎症细胞，以巨噬细胞为主，未见到嗜酸性粒细胞。Ova组：气道上皮细胞增生肥大，上皮不完整，纤毛排列紊乱，上皮下基底层增厚，平滑肌肥大增生，气道和血管周围、肺泡隔内见大量嗜酸性粒细胞浸润。Anti—CCR3组：与Ova组相比，气道和血管周围炎症明显减低。IgG组：与Ova组相比，气道和血管周围炎症未见明显减少。6．抗CCR3抗体(PBND03)对Ova致敏和激发后导致的粘液高分泌的影响PAS染色发现：Saline组：气道上皮完整，几乎无杯状细胞增生和粘液形成。Ova组：气道上皮杯状细胞增生肥大，并有粘液形成，上皮脱落不完整。Anti—CCR3组：与Ova组相比，气道粘液分泌明显减低。IgG组：与Ova组相比，粘液分泌未见明显减少。用粘液的储备指数(MOR)和杯状细胞的化生指数(HR)来定量分析气道粘液的形成，结果发现Ova组与Saline组相比较MOR和HR均明显增高。抗CCR3抗体可明显减低Ova导致的MOR和HR增高，而非特异性IgG对Ova导致的MOR和HR增高无抑制作用。7．抗CCR3抗体(PBND03)对Ova致敏和激发后导致气道粘液素基因MUC5AC mRNA的表达的影响如图6所示，Saline组于MUC5AC位置没有出现条带，而Ova组于MUC5AC位置出现明显条带。和Ova组相比较，抗CCR3组在MUC5AC部位没有出现明显的条带。IgG组在MUC5AC部位出现的条带和Ova组相似。上述结果说明Ova致敏和激发引起了气道粘液素基因MUC5AC mRNA的表达的增高，而抗CCR3抗体抑制了Ova导致气道粘液素基因MUC5AC mRNA表达的增高。8．抗CCR3抗体(PBND03)对BALF中细胞因子水平的影响Ova组中IL-4较Saline组明显增高，而抗CCR3组中的IL-4较Ova组明显下降。Ova组中的IFN-γ水平和Saline组相比明显下降，但是和Ova组相比，抗CCR3组中的IFN-γ水平没有明显变化。IgG组中无论是IL-4还是IFN-γ水平与Ova组相比均无明显变化。上述结果表明，抗CCR3抗体减少了Ova所致的肺部IL-4的水平增高，而对IFN-γ水平却没有影响。结论1．本研究合成了CCR3细胞外区NH2端多肽，并以此多肽免疫小鼠成功地获得了鼠抗人抗CCR3抗体。2．本研究合成的鼠抗人抗CCR3抗体可显著抑制哮喘小鼠的气道炎症和粘液高分泌等哮喘发病的特征性病理改变，其作用与直接阻断炎症细胞浸润和对IL-4下调有关。3．本研究结果提示阻断CCR3通路是有效的治疗哮喘的途径，为进一步研究开发相关的哮喘治疗药物提供了实验参考。更多还原

【Abstract】 IntroductionAsthma is an inflammatory disease in respiratory system, which is characterized by airway inflammations, hyperresponsiveness （AHR）. Clinical study and mouse model of asthma showed leukocytes were involved in the pathophysiologic process of asthma that eosinophil are found to play key role in the disease, due to the toxic granular proteins they secrete and the membrane products. In response to the chemokines generated locally, eosinophils from the microcirculation accumulate in the airway and result in pulmonary inflammation.Many small molecular proteins （8-10 kDa） have been identified and characterized as chemokines by their actions on distinct subtypes of leukocytes. Chemokines regulate immune cells by binding to 7-transmenbrane G protein-coupled receptors including CCR1, CCR2 and CCR3 et al. The distribution and expression of these receptors vary in distinct cells.CCR3 is a major chemokine receptor expressed on the surface of eosinophil, which is responsible for the cells activation and chemotaxis. In addition to eosinophil, CCR3 also exist in basophil, mast cell, and Th2 cell. In vitro and vivo studies, several C-C types of chemokines are shown to be active on eosinophil in allergic inflammation. Chemokines that are selective for CCR3 include eotaxin, eotaxin-2, and eotaxin-3, whereas RANTES, MCP-3, and MCP-4 bind to other chemokine receptors other than CCR3^[11]. The chemokines that contribute to aberrant eosinophil recruitment to the airways in asthma are not certain. It may be that different chemokines, operate in the process of asthma by different immune mechanisms. It was reported that the binding of an oligodeoxynucleotide, so-called anti-sense, to thecomplementary sequence of a messenger RNA can prevent the synthesis of the encoded protein. So making the blockade of a common receptor is more appealing. CCR3 have been identified and characterized from other species, such as mice, rat, guinea pig and non-human primates, and antibody of CCR3 are generated to aim at this attractive biological target for therapeutic intervention.The treatment of asthma has been improved by the implementation of management guideline in recent years, with further development in the study for the mechanism of asthma. Many medicines such as corticosteroids, theophylline etc. have been used to control asthma as anti-inflammatory agents. However, the effects of those drugs are not satisfied in the clinical practice. Therefore, a new agent for asthma treatment is required anxiously. For these reasons, CCR3 has been more important as a significant anti-inflammatory pharmacological target, and CCR3 antagonists are currently being developed for the treatment of asthma and other allergic disorders. Administration of an anti-CCR3 monoclonal antibody （mAb） was capable of inducing a targeted reduction of eosinophils in peripheral blood. Inhibitors of CCR3 showed these agents were effective on inhibiting eosinophil recruitment in allergen models of asthma. We also reported anti-mouse CCR3 mAb inhibited airway inflammation in mice^[18]. However there is no report to evaluate the effects of anti-human CCR3 antibody on airway inflammation, and mucus secretion in the mice model of asthma.Objective The purpose of the study is to generated murine mAb against the humanCCR3, using the 30-amino acid synthetic peptide which is the NH2-terminal of CCR3 as the immunizing antigen. Furthermore, we want to investigate the therapeutic effects of this specific antibody study in an athmatic mice sensitized and challenged with ovalbumin.Methods A peptide was synthesized, corresponding to the predicted NH2 terminus of the human CCR3 amino acid sequence. The peptide conjugated to Keyhole limpethemocyanin （KLH） was injected into BALB/c mice at 6-day intervals. Mice sera were tested by ELISA and the spleen cells of mouse that produced the most potent serum titer larger than 1: 6250 were used for fusion with SP2/0 cells. Positive hybridoma clones were selected by enzyme-linked immunosorbent assay. Hybridoma clones producing anti-human CCR3 mAbs were injected into mice for ascites production. The IgG fraction from the ascites fluid was purified and dialyzed. Western blot analysis was performed to identify the specificity of Mouse Anti-human CCR3 mAb. In Vivo study to determine the inhibitory effect of the antibody, mice were sensitized on the d 0 and d 14 by ip injection of ovalbumin （Ova） mixed with aluminum. At d 24, 25, and 26, mice were challenged by aerosolized 1% Ova in saline for 40 min. 32 mice were divided into 4 groups （8 mice each group）, Saline group, Ova group, Anti-CCR3 group, and IgG group. Antibody was administered by ip injection before OVA challenge at d 24, d 25, d 26 at the dose of 3 mg per kg, and the non-specific IgG were administered at the same time point in IgG group. The bronchoalveolar lavage fluid （BALF）, histopathology, mucus secretion were examined.Results:Generation and identification of the anti-human CCR3 mAbEnzyme-linked immunosorbent assay was employed to detect the affinity of the generated antibody. Antibodies from three different hybridoma clones, which are PBND03, PBND04, and PBND05 show high affinity with the peptide. The affinities are PBND03>1.28×l0⁹, PBND04≥2×10⁷, PBND05≥3.2×10⁸ respectively. To further identify the antibody we produced, those three antibodies were chosen to perform western-blot as the primary antibody. Lysate of Daudi cell expressing CCR3 was used to determine the specificity of the generated antibody to CCR3. As shown in Fig.1, remarkable strips were observed. The antibody from PBND03 clone was employed for further study in vivo because of its highest affinity.The anti-human CCR3 mAb decreased leukocyte in the BALF of allergic miceTo examine the effect of anti-human CCR3 mAb on inflammatory cells accumulation responding to allergen challenge, BALF was recovered. Total cells of BALF were counted and differentiated using morphological criteria. The total number of leukocytes and eosinophils in the Ova group was significantly greater than that in the Saline group. A comparison of individual cell numbers per ml of BALF reveals that these increases were mainly due to increases in eosinophil populations. There are no differences in total number of leukocytes and eosinophil between Ova group and IgG group. In comparison to Ova group, total number of leukocytes and eosinophils in anti-human CCR3 mAb groups was decreased significantly. No significant differences in neutrophil and lymphocyte were observed between the groups.The anti-human CCR3 mAb inhibited pulmonary inflammation in allergic miceTo determine if anti-human CCR3 mAb inhibit the airway inflammatory infiltration,we observed the pulmonary pathology stained with H&E of mice in all groups. Compared with mice treated with saline, exposure to Ova resulted in the development of a characteristic peribronchial and perivascular airway tissue infiltration. In comparison to mice in Ova group, no obvious change of pulmonary inflammation was observed in the mice administered with non-specific IgG. However, the allergic mice administered with anti-human CCR3 mAb exhibited a remarkable reduced inflammation in the lung tissue.The anti-human CCR3 mAb reduced mucus overprodution of allergic miceTo evaluate the inhibitory effect of anti-human CCR3 mAb on hypersecretion of mucus in airway, PAS stain was performed. In allergic mice, morphometric analyses of the PAS stained lung sections revealed obviously increase in mucus cells hypertrophy and in the percentage of airway mucus, which is described by HR and MOR respectively. Anti-human CCR3 mAb significantly reduced the mucus secretion. Compared with mice in Ovagroup, mice treated with anti-human CCR3 mAbexhibited decreased HR and MOR. No reduction of mucus hypersecretion induced by allergen was observed in mice treated with non-specific IgG mice.The anti-human CCR3 mAb decreased IL-4 in BALF of allergic miceTo determine the effect of anti-human CCR3 mAb on the pulmonary immune response in allergic mice, the concentration of IL-4, IFN-γ in BALF were measured. The IL-4 level in the Ova group was significantly greater than that in the Saline group. The mice treated with anti-human CCR3 mAb showed significant decrease of IL-4 in comparison to the allergic mice. The IFN-γ levels in Ova group was less than that in the Saline group and there were no differences between the anti-human CCR3 mAb groups and Ova group in IFN-γ levels. It was showed non-specific IgG had no effect on these cytokine levels in allergic mice.ConclusionWe found the antibody we generated was specific to CCR3. The allergic mice treated with the anti-human CCR3 antibody exhibited significant reduction of pulmonary inflammation and mucus overproduction accompanied by the alteration of cytokine in BALF. It is suggested the blockade of CCR3 is an appealing therapeutical target for asthma. The present research may provide an experimental basis for further study of this agent.更多还原