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。20世纪80年代后提出了哮喘的本质是气道慢性炎症和气道高反应性(airway hyperresponsiveness,AHR)。气道炎症是气道高反应性(AHR)的病理基础,黏液高分泌、气道高反应性、可逆性气流受限和气道重构是哮喘的重要特征,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,研究认为肺部EOS浸润是气道高反应性(AHR)发生的必要条件。
趋化素样因子1(chemokine-like factor 1,CKLFI),为一新基因超家族的趋化因子,是北京大学医学部免疫学系马大龙教授领导的实验室在国际上首先发现并成功克隆的一个新的具有趋化活性的细胞因子,获中国及国际专利,并命名为趋化素样因子1。CKLF1是一种分泌性蛋白,对CKLF1的体内外功能研究发现,大鼠CKLF1和人CKLF1相似,对中性粒细胞、单核细胞、淋巴细胞等具有明显的趋化活性,并具有刺激骨骼肌增殖活性和刺激骨髓细胞增殖活性,并且能与人CCR4结合,证实CKLF1是CCR4又一新的功能配体。CKLF1在活化的CD4~+/CD8~+T细胞表达上调。在注射CKLF1真核表达质粒的小鼠肺部炎性病变明显,与哮喘发病的病理改变非常相似,且正常人气道上皮细胞可表达CKLF1mRNA,其可因TNF-a的刺激而加强,肺组织的Western blot及免疫组化结果显示CKLF1基因在肺内表达显著。对进一步利用化学合成的CKLF1 C端肽C19、C27的体外功能研究,发现其对CCR4、CCR5转染细胞具有趋化作用,C19趋化活性较弱。CKLF1具有的炎细胞趋化活性及独特的致肺部病理改变等特征,我们便想到,CKLF1是否参与哮喘气道炎症及气道高反应性(AHR)的发生发展?由此本研究选择CKLF1的C端肽C19、C27在哮喘小鼠模型进行体内功能的研究,观察其对哮喘小鼠气道EOS炎症、气道黏液分泌及对气道反应性的影响。
研究目的在我们成熟的哮喘模型基础上,腹腔注射CKLF1的C端肽C19、C27,观察其对哮喘小鼠气道EOS炎症、气道黏液分泌及对气道反应性的影响。
研究方法选择健康雄性的C57BL/6小鼠,6—8周龄,体重18—22g。以卵白蛋白(OVA)作为抗原进行两次腹腔注射致敏和三次雾化吸入激发,建立哮喘模型,同时设立阴性对照组(以生理盐水(NS)代替OVA)。分别于激发前30min腹腔注射C19、C27不同剂量干预,分为:阴性对照组(NS组)、致敏攻击组(OVA组)、C19、C27不同剂量干预组。于最后一次抗原激发后24h检测支气管肺泡灌洗液(BALF)中炎症细胞,组织病理观察肺组织炎症和气道黏液分泌,并检测各组小鼠的气道反应性水平。分别以杯状细胞化生指数——指每mm长气管上皮中杯状细胞数目;上皮黏液储备指数——上皮过碘酸-雪呋(PAS)阳性染色面积比气管上皮总面积,作为气道黏液的定量观察指标。行肺组织病理切片观察嗜酸性粒细胞和其它炎症细胞的浸润。
研究结果
1 C19、C27干预对哮喘小鼠BALF中Eos数的影响
在OVA致敏攻击组,BALF中Eos数较阴性对照组明显增高(p<0.05)。C19、C27干预后,与OVA致敏攻击组比较,C19各剂量组Eos比例和Eos绝对数均有显著性差异(p<0.05),但C19各剂量组间无显著性差异;C27各剂量组Eos比例和Eos绝对数无显著性差异。
2 C19、C27干预对哮喘小鼠肺组织炎症浸润的影响
肺组织病理切片HE染色显示:阴性对照组小鼠气道及血管周围无Eos浸润,上皮完整;而OVA致敏攻击组小鼠气道及血管周围均可见大量炎症细胞和Eos浸润,并波及肺实质,上皮脱落不完整。C19干预显示各剂量组气道及血管周围仍可见一些炎症细胞,但Eos较OVA致敏攻击组明显降低,上皮基本完整。C27干预后较OVA致敏攻击组无明显改变。
PAS染色显示:阴性对照组气道未见GC增生和肥大,末见气道上皮细胞黏液分泌;OVA致敏攻击组可见气道上皮杯状细胞增生肥大,并有大量黏液分泌;C19、C27干预后镜下各组较OVA致敏攻击组无明显差异改变。
3 C19、C27干预对哮喘小鼠气道黏液的定量分析
OVA致敏攻击组和C19、C27干预组气道黏液的储备指数和杯状细胞的化生指数均高于阴性对照组(p<0.05),C19、C27干预各剂量组与OVA致敏攻击组比较差异无统计学意义。
4 C19、C27干预对哮喘小鼠气道反应性的影响
C19各干预组胸腔压(pleural pressure,Ppl)与OVA致敏攻击组比较显著降低(p<0.05),但C19各剂量组之间胸腔压差异无显著性;C27各干预组Ppl与OVA致敏攻击组比较并无显著性差异。
结论
1.CKLF1 C端肽C19可抑制哮喘小鼠气道Eos炎症,并能降低气道高反应性;C27对哮喘小鼠气道Eos炎症及气道高反应性没有明显影响。
2.CKLF1 C端肽C19、C27对哮喘小鼠的气道黏液高分泌无明显影响。
Background Asthma is a chronic airway 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 (Eos) 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.
The chemokine-like factor 1 (CKLF1) is a novel human cytokine isolated from PHA-stimulated U937 cells, which is firstly discoveried and cloned from the labortory of peking university center for human disease genomics. The CKLF1, which belongs to a novel gene family, has chemotaxis effect on different leukocytes both in vitro and in vivo. It can also stimulate proliferation of skeletal muscle cells and bone marrow cells. CKLF1 has a CC motif and the key amino acids around the motif are identical with those of TARC/CCL17 (Thymus- and activation-regulated chemokine) and
MDC/CCL22 (Macrophage Derived Chemokine), the cognate ligands for CCR4 which are up-regulated in the airways of atopic asthmatics. Thus, CKLF1 is a novel functional ligand for CCR4. CKLF1 is also found that it is up-regulated in activated CD4~+ and CD8~+ cells. A single intramuscular injection of CKLF1 plasmid DNA into BALB/c mice cause dramatic pathological changes in the lungs of treated mice. Those changes are similar to phenomena observed in asthma. C19 and C27 are C terminatio peptide of CKLF1, which are obtained by chemical synthesis. C19 and C27 are functional ligands for CCR4 and CCR5, they also exhibit chemotactic effect on leukocytes. Those data suggest that CKLF1 may play an important role in the pathogenesis of asthma. In this study we investigated the effects of intraperitoneal injection of C19 and C27 of C terminatio peptide of CKLF1 by different dosages on the airway inflammation and airway mucus secretion and airway hyperresponsiveness in a mouse model of asthma.
Objective Based on a mouse model of asthma we created previously, intraperitoneal injection of C19 and C27 of C terminatio peptide of CKLF1 by different dosages , then we examined the effects on the airway inflammation and airway mucus secretion and hyperresponsiveness.
Methods Male C57BL/6 mice, 6-8 week old, were sensitized two times and challenged three times by ovalbumin(OVA) as OVA/OVA group, and mice were treated by normal saline as control group. Before 30 minutes of every challenge, to intraperitoneal injection of C19 and C27 respectively by different dosages. So we assigned mice to four groups: Normal saline solution group(NS group), asthma model group(OVA group), C19 -treated groups, C27-treated groups. Then 24 hours after the last challenge, the bronchoalveolar lavage(BALF) inflammatory cell counts and the pathomorphological changes in the lung were analyzed; the goblet cell hyperplasia ratio(HR) and the epithelial cell mucus occupying ratio(MOR) were also measured; the level of airway responsiveness was detected.
Results:
1. The changes of Eos population in BALF after C19 and C27-treated.
We found Eos increased obviously in BALF after OVA challenge compared with NS group, p<0.05. Compared with OVA group, Eos in BALF was reduced significantly in C19-treated groups, p<0.05; but Eos in BALF was not reduced significantly by C27.
2. The change of pulmonary pathology after C19 and C27-treated.
In NS group mice, there were no Eos accumulation around airways and vessels, and no mucus secretion in the epithelial cells. However, in OVA group, we observed a significant increase in Eos infiltration around airways and vessels, and mucus hypersecretion in the epithelial cells. In C19-treated mice, there were little inflammatory cells around airways and vessels, however mucus hypersecretion has no change compared with OVA group. In C27-treated mice, both inflammatory cells and mucus hypersecretion have no change compared with OVA group.
3. Quantitative analysis of airways mucus after C19/C27-treated.
HR and MOR of C19-treated groups, C27-treated groups and OVA group increased significantly compared with NS group, p<0.05; but HR and MOR of C19-treated groups and C27-treated groups were still not significant difference compared with OVA group.
4. The change of airway responsiveness after C19 and C27-treated.
Pleural pressure (Ppl) of C19-treated groups were reduced significantly compared with OVA group, p<0.05; but Ppl of C27-treated groups were not significant difference compared with OVA group.
Conclusion
1. Eos in BALF can be reduced in C19-treated groups, C19 peptide can inhibit the airway inflammation and hyperresponsiveness; but C27 peptide has no effect on the airway inflammation and hyperresponsiveness.
2. Both C19 peptide and C27 peptide have no effect on the airway mucus hypersecretion.
引文
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