【作者】 刘莹；

【导师】 王秀芳；

【作者基本信息】 郑州大学 ， 儿科学， 2012， 硕士

【摘要】 对5岁以下的儿童这一特殊群体来讲,能引起喘息的疾病种类原因颇多,如何鉴别是困扰儿科呼吸专业医生的难题。研究结果表明有约40%的支气管哮喘及反复喘息患儿第一次喘息发生在一岁以内。国内、国外的许多研究资料证明,早期、规范的对儿童哮喘进行干预及管理,将有效改善疾病预后、提高患者生活质量并降低社会医疗负担。目前有越来越多的研究试图找到一种确切的实验室指标,用以从小于5岁的喘息儿童中鉴别出真正的支气管哮喘病人,但目前这个问题尚未解决。目的本临床研究对IL-10基因转录启动子的单核苷酸多态性(single-nucleotide polymorphisms,SNPs)进行研究,同时观察外周血IL-10、IgE及EOS水平,了解反复喘息患儿发作时体内变化,尤其是结合哮喘预测指数的不同与正常儿童进行比较,以探讨IL-10与5岁以下儿童反复喘息发病机制之间的关系。材料和方法本实验设计有喘息组与对照组。选择2010年12月-2011年4月在郑州大学第三附属医院儿内科门诊就诊或收入病房治疗的喘息患儿共152例入选喘息组,喘息患儿均为中国河南省汉族儿童,均处于急性发作期,年龄小于或等于5岁。入选患儿在最近1年内至少发作4次喘息或最近半年内已至少发作2次喘息,排除早产儿、先天性心脏病儿童、气道发育畸形儿童、足月小样儿。根据患儿本人有医师诊断的变应性疾病(例如变应性鼻炎、湿疹、食物过敏、变应性皮炎)或患儿父母任一方有医师诊断的哮喘病史,作为特应质高危因素。根据研究对象是否具有高危因素将喘息患儿又分为2组,具有高危因素之一的患儿入选喘息Ⅰ组(n=78；男56例,女22例),不具有高危因素的患儿入选喘息Ⅱ组(n=74；男52例,女22例)。随机选择同时期本院儿童保健部健康体检儿童46例(n=46；男26例,女20例)入选健康对照组,对照组儿童均排除特应质高危因素。所有研究对象于近2周内均未患感染性疾病、未应用过激素类药物及其他免疫调节药物,所有入组儿童均为足月出生儿童,3组患儿年龄、性别进行比较,均无统计学差异(P>0.05),入组前均告知其监护人并征得其知情同意。采用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)方法检测IL-10启动子-592位点A/C基因多态性,比较三组研究对象IL-10启动子-592位点的基因型频率及等位基因频率。采用酶联免疫吸附实验法(ELISA)对3组患儿外周血IL-10及总IgE水平进行定量测定。采用伊红染色法计数外周血EOS。对三组患儿的外周血IL-10、总IgE及EOS计数水平的均值进行比较,并进一步分析有无相关性。结果1.三组中均可检测到CC、AC、AA3种基因型,并且三组均以杂合子AC型比率较高,野生型CC型比率次之,纯合突变型AA型比率较小。其中喘息Ⅰ组CC、AC、AA基因型频率为25.6%、59.0%、15.4%,A、C等位基因频率为44.9%、55.1%；喘息Ⅱ组CC、AC、AA基因型频率分别为21.6%、63.5%、14.9%,A、C等位基因频率为46.6%、53.4%；对照组CC、AC、AA基因型频率为37.0%、52.2%、10.8%,A、C等位基因频率为37%、63%。三组间比较各基因型频率及等位基因频率分布差异均无统计学意义(P值均>0.05)。2.喘息Ⅰ组外周血IL-10、总IgE水平及EOS计数水平均高于喘息Ⅱ组及对照组(P值均<0.01),差异有统计学意义；喘息Ⅱ组外周血IL-10、总IgE水平均高于对照组(P值均<0.01),差异有统计学意义,喘息Ⅱ组EOS计数水平与对照组比较,差异无统计学意义(P值>0.05)。3.相关性分析显示喘息Ⅰ组外周血IL-10水平与IgE水平呈正相关关系(r=0.815,P<0.01),喘息Ⅱ组及对照组IL-10与EOS,IL-10与IgE均无相关。结论IL-10基因启动子-592位点基因多态性与5岁及其以下儿童反复喘息性疾病无相关性,但外周血IL-10水平与特应质及机体过敏因素存在高度相关,IL-10与具有特应质的5岁以下反复喘息儿童发病相关。更多还原

【Abstract】 For this special group of children under5years of age, there are many types of disease which can cause wheezing, and identifing these diseases is the difficult problem that distress the pediatric respiratory professional physicians. The results show that about40%children who under1year of age develop bronchial asthma and recurrent wheezing at the first time. Domestic and foreign research data show that the early specific intervention and management for children with asthma, will greatly improve the prognosis of diseases, inhance the quality of life and reduce the burden of social health. There are more and more researches trying to find an exact laboratory parameters to identify the true bronchial asthma from under5-year-old wheezing children, but they has yet to discover effective biological indicators.ObjectiveThe clinical study attempts to explore single nucleotide polymorphisms (single-nucleotide polymorphisms, SNPs) of IL-10gene transcription promoter, to observe the characteristic of the plasma concentration of IL-10, IgE levels and the peripheral blood eosinophil count (PBEC) in reccurent wheezing children under five years old, with the purpose of understandingin the immunological changes in their body. And what is more important is that the concept of asthma predictive index were introduced to compare wheezing children and normal children. Thus,we could study the relationship between IL-10and the pathogenesis of childhood wheezing.Materials and methodsThe experiment designed two groups:wheezing group(for152cases) and the control group(for46cases). Subjects of the wheezing group are all from hospital pediatric clinc or hospitalized patients from December2010to April2010.All wheezing children are Han nationality in China’s Henan Province and in acute stage, all of whom were wheezing at least four times in recent1year or at least two times in recent six months, excluding those with congenital heart disease, preterm infants,and small for gestational age.According whether the children have a physician-diagnosed allergic disease (including allergic rhinitis, eczema, food allergies, allergic dermatitis), or either/both of their parents have a history of physician-diagnosed asthma as the risk factors for atopic quality standards, we divide the wheezing group into two parts, children with one of the risk factors are wheezing group Ⅰ (for78cases,56males and22females), and children without risk factors are wheezing group Ⅱ ((for74cases,52males and22females).Healthy children (for46cases,26males and20females) who come from hospital children’s health care as the control group for the same period,, and the atopic qualitative risk factors were excluded in this group. All children inrolled were without history of infectious diseases, without history of steroid use and other immunomodulatory agent history in nearly two weeks.All children were full-term births,3groups were were not any significant different (P>0.05) in age and gender, and their parents informed consent.By using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), we detected polymorphism of IL-10gene-592A/C, and compared the frequency of genotypes and alleles frequency of IL-10gene-592A/C loci in three groups.The plasma concentration of IL-10, total IgE and PBEC were detected by enzyme-linked immonosorbent assay(ELISA),we used eosin staining method.to determine the PBEC count.The IL-10, total IgE and PBEC in three groups were compared, and the correlation were analysed.Results1. IL-10promoter-592A/C gene polymorphism was present in each group.There were three kinds of genotypes of the gene polymorphism.The frequency of each genotype of wheezing group Ⅰ was CC、AC、AA(25.6%,59.0%,15.4%) respectively, A and C allele frequency was44.9%,55.1%; the frequency of each genotype of wheezing group Ⅱ was CC、AC、AA (21.6%,63.5%,14.9%) respectively, A and C allele frequency was46.6%,53.4%; the frequency of each genotype of control group was CC, AC, AA (37.0%,52.2%,10.8%) respectively, A and C allele frequency was37%,63%..There was no significant difference in three groups(P>0.05) for the genotype and allele frequency.2. The serum levels of IL-10, TIgE and PBEC in wheezing group Ⅰ were higher than the group Ⅱ(P<0.01)and the control group (P<0.01),and the levels of IL-10, TIgE in wheezing group Ⅱ were higher than the control group (P<0.01), PBEC between the group Ⅱ and the control group have no difference(P>0.05).3. Correlation analysis shows that the serum of IL-10levels and IgE levels were positively correlated relationship (r=0.815, P<0.01).ConclusionsIL-10promoter-592A/C loci genetic polymorphism is not correlated with recurrent wheezing which often occured in children under5years old. But the serum levelof IL-10was correlated with atopy.更多还原