【作者】 李小波；

【导师】 沈华洁；

【作者基本信息】 浙江大学 ， 内科学， 2006， 硕士

【摘要】 研究背景 支气管哮喘(简称哮喘)是由多种细胞(如嗜酸性粒细胞、肥大细胞、T淋巴细胞、中性粒细胞、气道上皮细胞等)和细胞组分参与的气道慢性炎症性疾病。其中嗜酸性粒细胞(Eos)在哮喘的发病中起了重要的作用,嗜酸性粒细胞在气道局部的浸润是哮喘的最显著的特征之一。Eos来源于骨髓CD34~+祖细胞,哮喘小鼠经抗原激发后发生Eos有规律的自骨髓、经血液循环向气道迁移的过程。目前研究表明,哮喘患者接触过敏原后活化的Th2细胞分泌大量细胞因子和趋化因子,在IL-5、eotaxin等因子作用下,骨髓CD34~+祖细胞增殖、分化成熟为Eos并迁移至外周血和支气管黏膜,另一方面,骨髓CD34~+祖细胞本身也可迁移至外周血和支气管黏膜,并可增殖分化产生更多的CD34~+祖细胞,在局部旁分泌和自分泌的IL-5等细胞因子作用下,CD34~+祖细胞增殖、分化为Eos,成为局部Eos的重要来源之一。有研究发现,致敏小鼠经过敏原激发后,外周血及BALF中CD34~+祖细胞明显增加。哮喘患者吸入过敏原后诱导痰中CD34~+及CD34~+IL-5Rα~+细胞数明显增加,并且Eos数与CD34~+IL-5Rα~+细胞数明显正相关。 Eotaxin是一种Eos特异性的趋化因子,可与Eos表面的CCR3受体相结合,参与Eos从局部微循环趋化募集到炎症组织的过程。CCR3是eoatxin唯一的受体,其在Eos、Th2淋巴细胞、嗜碱性粒细胞及巨噬细胞都有表达,但CCR3优先在Eos上表达,是Eos主要趋化因子受体。近来研究表明,人骨髓CD34~+祖细胞表面存在CCR3受体,且哮喘病人接触变应原后CD34~+祖细胞表面CCR3表达增加,提示eotaxin/CCR3不仅在Eos的趋化中扮演重要角色也可能在CD34~+祖细胞的趋化和分化中起重要作用。由此我们建立哮喘模型,拟观察CD34~+祖细胞、Eos的更多还原

【Abstract】 Background Asthma is a chronic airways inflammatory disorder in which many cells(eosinophil,mast,T lymphocyte,neutrophil and airway epithelium) and cytokines participate associated with variable airflow limitation, airway mucus hypersecretion and airway hyperresponsiveness(AHR).Increasing evidence suggests that eosinophils play a central role in the pathogenesis of asthma.Eosinophils develop from CD34~+ hematopoietic progenitor cells. After exposure to allergen, actived TH2 lymphocytes in the airways secret sereval chemokines and chemoattractants.With the effect of IL-5 and eotaxin,CD34~+ progenitor cells multiply and differentiate to mature eosinophils which traffic from bone marrow (BM) into airway mucosa via the circulation, besides, CD34~+ progenitors itself also can migrate to circulation and airway mucosa derectly, as a important local resource of eosinophil with the effect of IL-5 and other chemokines. These CD34~+ progenitor not only can complete their differentiation into eosinophils but also have the capacity to produce eosinophil colonies, proving their ability to multiply. Increased number of CD34~+ cells in PB and airway in mice asthma model has previously been documented, after allergen exposure. Dorman and coworkers have examined, after challenged, there was significant increase of CD34~+ and CD34~+ IL-5R~+ cells in induced sputum from allergic subjects with asthma, further, the number of CD34~+ IL-5R~+ cells was closelyexpression on CD34+ progenitors significantly increases in asthmatics.These findingssuggest, CCR3/eotaxin is involved not only in the migration of eosinophil but also inthe migration and differentiation of CD34+progenitor. So we observe the kinetics ofCD34+progenitors and eosinophils in mice asthma model, in order to investigate theeffect of CCR3/eotaxin on the migration of eosinophils and CD34+progenitors.Objective Based on our mature mouse asthma model,the primery objective of thisstudy was to conduct a detailed kinetics characterzation of CD34+progenitors andeosinophils in bronchoalveolar lavage fluid,peripheral blood and bone marrow,toinvestigate the relationship of the kinetics with the expression of CCR3/eotaxin,moreimportantly, to investigate relationship of CD34+cells and CCR3/eotaxin with airwaysinflammation in asthma.Methods Male C57B1V6 mice,6-8 week old,were sensitized two times andchallenged three times by ovalbumin(OVA) as OVA/OVA group, and mice weretreated by normal saline as control group.Then BALF, BM, and PB was collected at6h,12h,24h,48h after the final allergen challenge respectively.Eosinophil and CD34+progenitor populations was determined,eotaxin was measured by ELISA.To showeosinophils infiltration,the histology of lung tissues was also observed.The expressionof eotaxin mRNA and CCR3 mRNA in the lung were measured by reversetrancription-polymerase chain reaction( RT-PCR).Results:1. The changes of pulmonary pathology and cells in BALF, BM and PB1.1.Pulmonary pathology shows, in group control, there are no eosinophils infiltrationaround bronchi and pulmonary vessels;however, in group model, we obserbed asignificant increase in eosinophils infiltration around bronchi and vessels beginning at6h after antigen challenge and reaching a peak at 48h.1.2.In the BALF,the eosinphilia was fist detected at 6h after antigen challenge and thatwas remained to 48h.1.3. In the PB, a significant increase in the eosinophil numbers was fist detected at24h after antigen challenge,that was remained to 48h.1.4. In the BM, a significant increase in the eosinophil numbers was also detected at 24h and 48h after antigen challenge.2. The changes of CD34+ progenitor and CD34+/CCR3+ cell in BALF,BM and PB2.1 In the BALF,there was a remarkable increase in the ratio and absolute number of CD34+ progenitor at 12h 24h and 48h. There was not significant difference in the expression of CCR3 in CD34+ progenitors,but the absolute number of CD34+/CCR3+ cell inreased remarkablely from 12h to 48h after antigen challenge.2.2 In the PB, we did not detect any significant difference in the ratio of CD34+ progenitor, and there was not any significant increase in the absolute number of CD34+progenitor until 48h. the expression of CCR3 in CD34+progenitor was elevated remarkablely from 12h to 24h.The absolute number of CD34+/CCR3+cell was also elevated from 12h,but it remained to 48h.2.3 In the BM, we did not detect any signigicant changes in the ratio of CD34+ progenitor.however, there was a significant increase of absolute number of CD34+ progenitor at 48h. The expression of CCR3 on CD34+progenitor was increased remarkablely at 12h and 24h, but the absolute number of CD347CCR3+cellsinreased remarkablely from 12h to 48h.3. The changes of eotaxin in BALF,BM and Serum3.1 In the BALF,the eotaxin levels increased at 6h after antigen challenge,this wasfollowed by a decrease,but the signigicant difference was remained to 24h, the eotaxin returned to baseline level at 48h.3.2 Serum eotaxin levels fluctuated after antigen challenge,but never exceeded those observed in control groups.3.3 We detected a slight increase in BM eotaxin level,but there was never sigificant difference between model and control groups at any time points.4. The expression of eotaxin mRNA and CCR3 mRNA in the lungCompared with control group,the expression of eotaxin mRNA and CCR3 mRNA inthe lung were increased remarkablely in all time points.5. The correlative analysis between the number of Eos in BALF and other detected parametersobserved in control groups.3.3 We detected a slight increase in BM eotaxin level,but there was never sigificantdifference between model and control groups at any time points.4. The expression of eotaxin mRNA and CCR3 mRNA in the lungCompared with control group,the expression of eotaxin mRNA and CCR3 mRNA inthe lung were increased remarkablely in all time points.5. The correlative analysis between the number of Eos in BALF and other detected parametersThe number of Eos in BALF was closely correlated with the number of Eos and the ratio of CCR3+ in CD34+progenitors and the number of CD34+/CCR3+cells in the BM,however,there was not significant correlation between the number of Eos in BALF and the number of CD34+progenitors in the BM. Conclusion1. The expression of CCR3 on CD34+progenitors was up-regulated in BM of asthma mice,which may favor the eosinophilia in PB and the infiltration of eosinophils in asthma airways and vessels.2. CCR3/eotaxin is involved in the migration of CD34+progenitors and it is also involved in the infiltration of eosinophils in asthma airways.更多还原