基于小鼠嗜碱性粒细胞活化致敏性模型的建立

Establishment of sensitization model based on the activation of basophil in mice

目的研究BALB/c致敏小鼠外周血中嗜碱性粒细胞表面分子CD63表达情况,构建小鼠嗜碱性粒细胞激活试验(BAT)模型以评价小分子药物(LMWC)致敏性。方法以流式细胞术(CD45 APC^(low)/IgE FITC^(high))和瑞氏-姬姆萨染色血涂片检测对照组和卵清蛋白(OVA)致敏小鼠(OVA致敏组)嗜碱性粒细胞的形态和数量;以CD45 APC^(low)/IgE FITC^(high)作为鉴定小鼠嗜碱性粒细胞的流式方案,anti-IgE为阳性对照体外与小鼠嗜碱性粒细胞孵育,观察CD63的表达,建立小鼠BAT模型;以青霉素G和BSA的耦联物(PG/BSA)致敏小鼠(PG/BSA致敏组),青霉素G和OVA的耦联物(PG/OVA)体外激发,验证模型的有效性。结果与对照组小鼠相比,OVA致敏组小鼠嗜碱性粒细胞的形态和数量差异无统计学意义(P=0.655,P=0.667);anti-IgE体外可刺激小鼠嗜碱性粒细胞表面CD63的表达;OVA致敏组小鼠外周血体外与OVA孵育后,CD63表达高于对照组[(59.70±37.93)%vs(2.15±1.13)%,差异有统计学意义(P=0.016);PG/BSA致敏组小鼠的外周血体外与PG/OVA孵育后,CD63表达高于对照组[(62.74±41.05)%vs(2.73±1.40)%],差异有统计学意义(P=0.019)。结论特定致敏原体外可引起已致敏小鼠的CD63表达上升,表明成功构建了小鼠BAT模型,并以PG验证了此模型的有效性,其可用于评价LMWC的致敏性。

ObjectiveTo detect the expression of CD63 of basophil in the peripheral blood of sensitized BALB/c mice,and to establish the basophils activation test(BAT)for assessing the sensitization of low molecule w eight compounds(LM WC).Methods The morphology and number of basophils in control BALB/c mice(control group)and ovalbumin(OVA)-sensitized BALB/c mice(OVA-sensitized group)w ere determined by flow cytometry(CD45APClow/IgE FITChigh)and peripheral blood smears stained by Wright-Giemsa.CD45 APClow/IgE FITChighw as used as the flow scheme to identify mice basophils,and anti-IgE w as used as a positive control to incubate w ith basophils in vitro to observe the expression of CD63 and establish the BAT of mice.In addition,the BALB/c mice w as sensitized w ith penicillin G conjugated BSA(PG/BSA group),and the blood w as stimulated by penicillin G-conjugated OVA(PG/OVA)in vitro to observe the expression of CD63,thereby detect the effectiveness of BAT.ResultsFlow cytometry and blood smears show ed that the morphology and numbers of basophils in control group and OVA-sensitized group had no significant difference(P=0.655,P=0.667).Anti-IgE could stimulate the expression of CD63 on basophils.After the incubation w ith OVA in vitro,the CD63 expression in peripheral blood of the OVA-sensitized group w as higher than the control group[(59.70±37.93)%vs(2.15±1.13)%,P=0.016].After the incubation w ith PG/BSA in vitro,the CD63 expression in peripheral blood of the PG/BSA-sensitized group w as higher than the control group[(62.74±41.05)%vs(2.73±1.40)%,P=0.019].ConclusionSpecific allergen in vitro can lead to increased CD63expression in the sensitized mice,indicating the successful construction of BAT in mice,w hich is effective verified by PG and can be used to evaluate the sensitization of LM WC.