【作者】 高琦；

【导师】 孙桂香；

【作者基本信息】 天津医科大学 ， 儿科学， 2008， 硕士

【摘要】 目的1型糖尿病是儿童常见的内分泌疾病,它的发病是遗传,环境,免疫因素共同作用的结果。1型糖尿病是多基因疾病,白细胞介素6(IL-6)是近年研究认为可能与1型糖尿病相关的细胞因子。本研究检测1型糖尿病病例组及对照组外周血白细胞介素6的水平,探讨它在1型糖尿病患儿体内的变化,同时应用聚合酶链式反应-限制性片段长度多态性法(PCR-RFLP)对白细胞介素6基因启动子-572位点进行研究,探讨此位点基因多态性是否与1型糖尿病相关,对于不同基因型进一步探讨其是否与外周血IL-6的水平,发病年龄及发病时空腹C肽水平相关。方法1应用放射免疫分析法检测1型糖尿病患儿组及对照组外周血IL-6水平。2应用盐析法提取病例组与对照组外周血的DNA,并在1%琼脂糖凝胶电泳上观察提取结果。3应用聚合酶链式反应对病例组及对照组包含IL-6基因启动子-572位点的特异片段进行扩增,扩增的目的片段用2.5%琼脂糖凝胶电泳检测。4将扩增产物用MbiⅠ内切酶进行酶切,酶切产物在3%琼脂糖凝胶电泳上检测,并对其进行定性分析。5应用放射免疫分析法检测病例组外周血的空腹C肽水平。6根据Hardy-Weinburg遗传平衡定律检查病例组及对照组所得基因型是否具有群体代表性。7应用SPSS15.0统计软件对所得数据进行分析,其中病例组与对照组外周血IL-6水平的比较及病例组中首诊病人与复诊病人外周血IL-6水平的比较均采用两组独立样本的t检验。病例组与对照组的IL-6基因启动子-572位点基因型频率的比较采用Fisher精确概率法,等位基因频率的比较采用x~2检验,用相对比值比及95%可信区间表示相对危险度。不同国家IL-6基因启动子-572位点基因型频率的比较采用x~2检验或Fisher精确概率法。病例组不同基因型间IL-6水平,发病年龄及发病时空腹C肽水平的比较均采用两组独立样本的t检验。结果1病例组与对照组儿童性别及年龄的差异无统计学意义(P＞0.05),具有可比性。2病例组与对照组儿童外周血IL-6水平的比较差异无统计学意义,病例组中首诊病人与复诊病人外周血IL-6水平的比较差异仍无统计学意义。(P＞0.05)3 IL-6基因启动子-572位点经PCR扩增后产物片段为163bp,酶切后分为3种基因型,CC型为1条带(163bp),GC型为3条带(163bp,101bp,62bp),GG型为2条带(101bp,62bp)。4病例组与对照组基因型频率符合Hardy-Weinburg遗传平衡定律,具有群体代表性。5对照组IL-6基因启动子-572位点基因型及等位基因频率的分布与中国其他研究结果比较差异无统计学意义(P＞0.05),与日本比较差异无统计学意义(P＞0.05),与法国、美国比较差异有统计学意义(P＜0.05)。6病例组与对照组IL-6基因启动子-572位点基因型频率及等位基因频率分布的差异均有统计学意义(P＜0.05),等位基因频率的相对风险分析示G等位基因的携带者患1型糖尿病的风险是C等位基因携带者的1.939倍(OR=1.939,95%CI=1.023～3.678)。7病例组IL-6基因启动子-572位点不同基因型间IL-6水平的差异无统计学意义。(P＞0.05)8病例组IL-6基因启动子-572位点不同基因型间发病年龄的差异有统计学意义(P＜0.05),其中携带G等位基因的患儿发病年龄早于CC基因型的患儿。9病例组IL-6基因启动子-572位点不同基因型间空腹C肽水平的差异无统计学意义。(P＞0.05)结论11型糖尿病患儿体内IL-6水平无变化,IL-6基因启动子-572位点不同基因型患儿体内的IL-6水平亦无变化。2不同种族IL-6基因启动子-572位点基因多态性存在差异,亚洲人群可能具有相似的基因型及等位基因分布,欧美国家可能具有相似的基因型及等位基因分布。3 IL-6基因启动子-572位点基因多态性可能与1型糖尿病的发病存在相关性,其中G等位基因可能是1型糖尿病的易感基因。4 IL-6基因启动子-572位点不同的基因型可能与患儿患病年龄存在相关性,其中GG+GC基因型的患儿的患病年龄早于CC基因型。5 IL-6基因启动子-572位点不同的基因型患儿患病时空腹C肽水平无差别。更多还原

【Abstract】 Objective Type 1 diabetes is a common disease of chinldren,The pathogenesis of type 1 diabetes involve genetically factors,environmental factors and immunological factors.Type 1 diabetes belongs to polygenic disease.IL-6 is considered to play a roal in the pathogenesis of type 1 diabetes,so we detected the level of IL-6 of patients and controls to study the change of IL-6 level in children of type 1 dianbetes,in the same time,we detected the genotype of IL-6-572 of patients and controls by Polymerase Chain Reaction-Restriction Fragment length Polymorphism(PCR-RFLP)to study the relationship between -572G/C polymorphism of IL-6 promoter and type 1 diabetes, we also studied the difference of IL-6 level,the onset year and the C peptide between different genotype of IL-6-572.Methods1 The level of IL-6 of patients and controls was detected by Radioimmunologic method.2 The DNA of patients and controls was extracted by salt fractionation and the purity of DNA was detected by 1.0%agarose gel electrophoresis.3 The promoter of IL-6-572 was amplificated by PCR and the amplification was detected by 2.5%agarose gel electrophoresis.4 The amplification was cut by incision enzyme MbiⅠand the genotype was detected by 3.0%agarose gel electrophoresis to undertake qualitative analysis.5 The C peptide of patients and controls was detected by Radioimmunologic method.6 The representation of genotype of patients and controls was detected accorrding to Hardy-Weinberg equilibrium.7 The data was analyzed by sofeware of spss15.0,The IL-6 level of patients and controls and the IL-6 level of preliminary diagnosed patients and re-examination diagnosed patients were all compared by two indepentent t test.The -572 genotype frequencies of IL-6 promoter of patients and controls were compared by Fisher exact probability test,the IL-6-572 allele frequencies were compared byχ~2 test and the relative risk was expressed by OR and 95%CI.The IL-6-572 disposition of different countries were compared byχ~2 test or Fisher exact probability test.The IL-6 level,the onset year,the C peptide of different genotype were also compared by two indepentent t test.Results1 There was no difference between patients and controls in spite of sex and age.2 The level of IL-6 was not different between patients and controls,and the level of IL-6 was also not different between the preliminary diagnosed patients and re-examined diagnosd patients.3 The amplification of IL-6 -572 was 163bp and there were three genotypes after cuting by incision enzyme,CC genotype was one bright bar(163bp)on the electropherogram,CG genotype was three bright bar(163bp,101bp,62bp)and GG genotype was two bright bar(101bp,62bp)on the electropherogram.4 The genotype and allele frequencies of IL-6-572 were all in Hardy-Weinburg equilibrium,so the patients and controls were representative.5 The IL-6-572 genotype and allele frequencies were not different between the controls and other Chinese people and Japanese,but which were different between the controls and France and American.6 The IL-6-572 genotype and allele frequencies were different between the patients and the controls(P＜0.05),the relative risk of allele of type 1 diabetes indicated G allele was 1.939 times to the G allele,odds ratio is 1.939,95%confidence interval is 1.023～3.678.(OR=1.939,95%CI=1.023～3.678)7 The comparison of the level of IL-6 between GG+GC genotype and CC genotype had no statistical significance.(P＞0.05)8 The comparison of the onset year between GG+GC genotype and CC genotype had statistical significance(P＜0.05),the onset year of child who take along G allele was earlier than the C allele.9 The comparison of C peptide between GG+GC genotype and CC genotype had no statistical significance.(P＞0.05)Conelusion(1)The level of IL-6 is not different between patients and controls,the IL-6 level is also not different between different genotypes of IL-6-572.(2)Different race has different frequency of IL-6-572 genotype and allele,The Asian have resemble frequency of IL-6-572 genotype and allele,and the European and American have resemble frequency.(3)IL-6-572 polymorphism is maybe associated with type 1 diabetes of children and the G allele maybe the predisposing gene of type 1 diabetes of children.(4)The different genotype of IL-6-572 is maybe associated with the onset year,the onset year of child who take along GG+GC genotype is earlier than the CC genotype.(5)The different genotype of IL-6-572 is not associated with C peptide.更多还原