小儿哮喘花剥苔产生的免疫机制研究

【关键词】  花剥苔;

    ［摘要］  目的：从免疫学角度探讨哮喘患儿花剥苔形成的机制。方法：采用免疫组化方法检测舌苔脱落上皮细胞中角蛋白13及bcl2蛋白的含量。应用流式细胞术检测外周血CD4+及细胞趋化因子受体3（cell chemokine receptor3, CCR3）的表达量。采用放射免疫法检测血清中皮质醇的含量。结果：哮喘花剥苔组患儿外周血CD4+和CCR3水平明显高于哮喘非花剥苔组及正常组（P＜0.05)；哮喘花剥苔组、非哮喘花剥苔组血清中皮质醇含量低于其余各组（P＜0.05)。哮喘花剥苔、非花剥苔组血清IgE水平明显高于其余各组（P＜0.05)。哮喘花剥苔组、非哮喘花剥苔组舌苔上皮脱落细胞中角蛋白13的含量明显低于哮喘非花剥苔组、非哮喘非花剥苔组和正常对照组（P＜0.05)。哮喘花剥苔组患儿舌苔上皮脱落细胞中bcl2蛋白含量与其余各组比较无统计学差异。结论：花剥苔的形成与免疫反应密切相关，血清皮质醇水平降低、CD4+及CCR3增多，可能促进了花剥苔的形成。同时，舌上皮细胞角化障碍及舌上皮细胞凋亡亦可能促进花剥苔的形成。

　　［关键词］  花剥苔; 哮喘; 免疫

　　Immunological mechanism of exfoliative tongue fur in children with asthma

　　ABSTRACT  Objective: To explore the immunological mechanism of exfoliative tongue fur in children with asthma.  Methods: Thirtynine children with asthma, twentyeight children with repetitive respiratory tract infection (nonasthma) and eleven healthy children were divided into five groups, which were asthma with exfoliative fur or with nonexfoliative fur groups, nonasthma with exfoliative fur or with nonexfoliative fur groups and normal control group. The concentrations of keratin 13 and bcl2 in cells exfoliated from tongue fur were detected by immunohistochemical method. The expression levels of blood cell chemokine receptor3 (CCR3) and CD4+ were examined by flow cytometry, and the levels of serum cortisol and IgE were detected by radioimmunoassay. Results: The levels of blood CD4+ and CCR3 of children with asthma and exfoliative fur were higher than those in the asthma with nonexfoliative fur group and the normal control group (P＜0.05). The serum level of cortisol in the groups of asthma with exfoliative fur and nonasthma with exfoliative fur were lower than that in the other groups (P<0.05). The serum levels of IgE in asthma with exfoliative fur or with nonexfoliative fur groups were higher than that in the other groups (P<0.05). Concentrations of keratin 13 in the cells exfoliated from tongue fur in the groups of asthma or nonasthma with exfoliative fur were lower than that of the other groups (P<0.05). There was no significant difference of expression level of bcl2 in the cells exfoliated from tongue fur among these five groups. Conclusion: There is a reasonably close relationship between the formation of exfoliative tongue fur and the immune system such as low level of serum cortisol and high levels of blood CD4+ and CCR3, which may all promote the formation of exfoliative fur. The disability of keratinization and apoptosis of epithelial cells of tongue may also be one cause for its formation.

　　KEY WORDS  exfoliative fur; asthma; immunity

　　花剥苔在中医辨证中具有十分重要的诊断意义。临床发现小儿多见花剥苔，尤其在哮喘患儿中发生率更高，且不易消除［1］。但是其发生原因尚不清楚，本文试从免疫学角度探讨哮喘患儿易出现花剥苔的机制。

　　1  资料与方法

　　1.1  主要试剂  IgE试剂盒，灵敏度为10 U/ml (24 ng/ml)，批号20021002，购于深圳晶美生物工程公司（美国BectonDickinson公司产品）；异硫氰酸荧光素标记细胞趋化因子受体3（cell chemokine receptor3, CCR3）试剂盒，批号FAB155P，购于RD公司；异硫氰酸荧光素CD4及相应同型对照试剂盒，批号FAB1325P，购自美国Coalter公司（法国Immunotech公司产品）；皮质醇试剂盒，批号20021001，由上海生物制品研究所提供；角蛋白13试剂盒，批号REV03012F，购于美国Neomarkers公司；bcl2蛋白试剂盒，批号REV012402M，购于美国Neomarkers公司。

　　1.2  主要仪器  EPICSXL流式细胞仪，美国Coalter公司产品；ELX800酶标仪，购于上海精密仪器有限公司；SN682放射免疫γ记数器，科学院上海原子核研究所日环仪器厂产品。

　　1.3  诊断标准  哮喘诊断标准根据全国儿科哮喘协作组制订的《儿童哮喘诊断标准和常规》［2］：（1）喘息呈反复发作者（或可追溯与某种变应原或刺激因素有关）；（2）发作时肺部闻及喘鸣音；（3）平喘药有明显疗效。花剥苔诊断标准：舌苔部分剥落，剥落处舌面光滑无苔者［3］。

　　1.4  病例选择与分组  2003年7月～2004年5月，上海中医药大学龙华门诊诊断为哮喘的患儿39例以及反复呼吸道易感儿童28例，其中男43例，女24例；年龄最小20个月，最大11岁，平均（5.0±2.3）岁。39例哮喘患儿分为哮喘花剥苔组（18例）和哮喘非花剥苔组（21例）；28例反复呼吸道易感儿童分为非哮喘花剥苔组（14例）和非哮喘非花剥苔组（14例）。所有患儿近2个月内均未予糖皮质激素治疗。另选择上海市徐汇区田林第三小学二年级健康无过敏史的小学生11名作为正常对照组。3组患儿年龄、性别比较无统计学差异。

　　1.5  检测指标  各组患儿空腹，清晨7:00至上海中医药大学曙光医院中心实验室免疫实验室，各采静脉血4 ml备检。（1）IgE检测：取血清，采用酶联免疫吸附测定法，用ELX800酶标仪检测光密度（optical density, OD）值；（2）皮质醇测定：取血清，采用放射免疫分析法，以相对结合率对浓度对数的标准曲线求得皮质醇浓度；（3）CCR3、CD4检测：取全血1 ml，用依地酸100 μl抗凝，双色免疫荧光标记，用流式细胞仪进行检测；（4）舌苔脱落细胞角蛋白13、bcl2表达量检测：用无菌小匙刮取舌中部舌苔，花剥苔者在剥处刮取，置入盛有1 ml生理盐水的Eppendorf管中，离心、涂片、固定，采用免疫组化3, 3’二氨基联苯胺染色，角蛋白13和bcl2在胞浆或胞膜上表达呈棕黄色，应用Motic数码医学图像分析系统进行半定量分析。

　　1.6  统计学方法  计量资料均数用x±s表示，采用单因素方差分析。