负载卵清蛋白的树突细胞体内回输对过敏性哮喘的影响

摘要

目的：探讨负载卵清蛋白（ OVA）的树突细胞回输至小鼠体内对过敏性哮喘的影响。方法将30只雄性BALB/c小鼠采用随机数字表法分为3组，每组10只。健康对照组不做任何处理；哮喘组给予磷酸盐缓冲液（PBS），然后致敏和激发；OVA-DC干预组尾静脉注射负载OVA的树突细胞1×106个，然后致敏和激发。将小鼠骨髓细胞用重组小鼠粒系-巨核系集落刺激因子（rmGM-CSF）和重组白介素4（rmIL-4）诱导分化为树突细胞，并通过形态学和流式细胞术检测细胞表面的CD11c予以鉴定。用OVA和脂多糖（ LPS）刺激后，流式细胞术检测细胞表面的主要组织相容性复合体Ⅱ类分子（ MHCⅡ）和CD86，以此判断树突细胞的成熟状态；混合淋巴细胞增殖试验（ MLR）检测不同状态的树突细胞刺激淋巴细胞增殖的能力。通过尾静脉注射树突细胞至小鼠体内，然后在特定时间进行致敏和激发，观察小鼠的生存状态、测定脾脏质量；激发后24 h收集小鼠肺泡灌洗液检测组胺水平；取肺组织行苏木素-伊红（ HE）染色，镜检观察肺部炎症严重程度。结果小鼠骨髓细胞经rmGM-CSF和IL-4诱导分化为树突细胞，树突细胞在负载OVA后免疫表型未发生明显的变化，仍处于未成熟状态，刺激淋巴细胞增殖的能力也很微弱。3组脾脏质量和肺泡灌洗液组胺水平比较，差异有统计学意义（ P<0.05），OVA-DC干预组较健康对照组和哮喘组升高，哮喘组较健康对照组升高（ P<0.05）。HE染色显示OVA-DC干预组小鼠肺组织气道管壁增厚，周围有大量炎性细胞浸润；哮喘组小鼠炎性反应较轻；健康对照组小鼠未发现炎性反应的特征。结论负载OVA后的树突细胞通过尾静脉回输至小鼠体内可以促进过敏性哮喘的发生，树突细胞在过敏性哮喘的发生机制方面发挥重要的作用。%Objective To investigate the effect of feedback of ovalbumin( OVA) - loaded dendritic cells( DCs) on mice with asthma. Methods Male BALB/c mice were divided into 3 groups by by random number table method,there were 10 mice in each group. The mice in healthy control group received no additional treatment;The mice in asthma group received PBS,and then received sensitization and challenge;The mice in OVA-DC treatment group were injected with OVA-loaded DCs(1 × 106 cells)through caudal vein,and then received sensitization and challenge. Bone marrow cells of mice were cultured in the presence of rmGM -CSF and rmIL-4,and differentiated to DCs. The cells were identified by morphology and surface marker CD11c using flow cytometry. After stimulated with OVA and LPS respectively,flow cytometry was used to detect MHCⅡand CD86 ,thus the mature status of DCs was determined. Mixed lymphocyte reaction( MLR)was performed to assess the ability of DCs in stimulating the proliferation of lymphocytes. Mice were injected with OVA-loaded DCs through caudal vein,followed by OVA stimulations and challenges at a specific time,survival condition of the mice was observed,and the mass of spleen was measured;24 h after challenges,the level of histamine in BALF was measured;After HE staining,the severity of inflammation in lung tissue was observed by microscope. Results Bone marrow cells of mice can be induced by rmGM-CSF and rmIL-4 , and differentiated into DCs,no apparent changes of immunophenotyping were observed in OVA -loaded DCs,OVA -loaded DCs remained immature,the ability of DCs in stimulating the proliferation of lymphocytes was weak. There were significant differ-ences in the mass of spleen and the level of histamine in BALF among 3 groups(P<0. 05),the above 2 indexes in OVA-DC treatment group were significantly higher than those in healthy control group and asthma group,the above 2 indexes in asthma group were significantly higher than those in healthy control group(P<0. 05). HE staining indicated that the lung tissue airway walls in OVA-DC treatment group became thicker,infiltration of inflammatory cells existed around airway walls. The inflammato-ry reaction was lighter in asthma group, the characteristic of inflammatory reaction was not found in healthy control group. Conclusion Feedback of OVA-loaded DCs through caudal vein can promote the occurrence of allergic asthma,DCs may play an important role in the occurrence mechanism of allergic asthma.