豚鼠哮喘模型单个核细胞及嗜酸细胞与肺微血管内皮细胞粘附性的研究

A study of adhesion of peripheral lymphocytes and eosinophils to lung capillary endothelial cells in a guinea pig asthma model

目的:观察哮喘豚鼠模型嗜酸细胞和淋巴细胞与肺微血管内皮细胞的粘附率,探讨肺微血管内皮细胞在哮喘模型发病中的功能变化及炎症细胞与其粘附的分子基础。方法:复制豚鼠哮喘模型;分离、培养和鉴定肺微血管内皮细胞;转染核因子(NF)-κB反义寡核苷酸;分别用对照组血清和模型组血清孵育;分离外周血淋巴细胞和嗜酸细胞;采用RT-PCR法测定内皮细胞和血管细胞粘附分子-1(VCAM-1)及嗜酸细胞趋化因子(eotaxin)mRNA的表达强度,EMSA法测定内皮细胞NF-κB、活化蛋白(AP-1)的DNA结合活性。结果:(1)肺微血管内皮细胞在模型组血清刺激下,与两组外周血淋巴细胞及仅与模型组嗜酸细胞的粘附率显著升高,内皮细胞与嗜酸细胞两者的激活,在嗜酸细胞粘附中起明显的协同作用;(2)肺微血管内皮细胞经模型组血清刺激后,VCAM-1和eotaxin mRNA表达量显著增多,NF-κB、AP-1的DNA结合活性也显著升高。结论:肺微血管内皮细胞可被哮喘模型血清激活,使粘附分子、趋化因子及核转录因子的合成和分泌增多,从而显著提高其与炎症细胞的粘附率,核转录因子NF-κB和AP-1对内皮细胞合成炎症因子可能起调控作用。淋巴细胞与嗜酸细胞的粘附机制不尽相同。

Objective:To investigate the adhesion rates of peripheral lymphocytes and eosinophils to lung capillary endotheoial cells in different culture conditions, and to observe the expression of VCAM-1 and eotaxin and the DNA binding activity of NF-Kb/AP-1 in lung capillary endothelial cells under the stimulation of serum in a guinea pig asthma model. Methods: (1)A guinea pig asthma model wsa reproduced, the peripheral lymphocytes and eosinophils were isolated. (2)The lung capillary endothelial cells of guinea pig were cultured and verified, and stimulated with serum of animals of asthma model. (3)The adhesion rates of lymphocytes and eosinophils to the cultured endothelial cell monolayers were measured. (4)The mRNA expressions of VCAM-1 and eotaxin in lung capillary endothelial cells were determined by of RT-PCR. (5)The DNA binding activities of NF-κB/AP-1 in endothelial cells were measured by electrophoretic mobility shifts assay (EMSA). Results: (1)The adhesion rates of lymphocytes and activated eosinophils to lung capillary endothelial cells stimulated with the serum of asthma model group were significantly elevated than those of controls (without stimulation). (2)The adhesion pattern of lymphocytes was different from that of eosinophils. (3)The rnRNA expressions of VCAM-1 and eotaxin in stimulated lung capillary endothelial cells were significantly elevated than those in controls. (4)The levels of DNA binding activity of NF-κB and AP-1 were significantly elevated in stimulated endothelial cells than those in controls. Conclusion:Lung capillary endothelial cells could be activated by the stimulation of serum in asthma group, and thus produced and secreted significantly increased amount of both adhesion molecules and chemotactic factors. The activation of lung capillary endothelial cells may contribute to the elevation of adhesion rates of inflammatory cells, which might play a synergistic action in eosinophil adhesion. The nuclear factor-κB and AP-1, as modulators, may accelerate the production of inflammatory cytokines in lung capillary endothelium.