【作者】 贾兴旺；

【导师】 田亚平；

【作者基本信息】 中国人民解放军军医进修学院 ， 临床检验诊断学， 2007， 硕士

【摘要】 目的为了研究IL-6基因多态性与心血管病的相关性，根据荧光共振能量转移和溶点曲线分析原理，建立了一种IL-6基因快速分型的实时荧光分析方法。并通过对中国北方地区汉族人IL-6基因启动子区-572G／C，-597G／A多态性与体重指数(BMI)和炎症因子等生化指标的相关性的调查，探索基因多态性与冠心病(CHD)发生发展的关系。方法利用实时荧光PCR方法检测我国北方地区汉族人群IL-6基因启动子-572G／C，-597G／A的多态性。甘油三脂(TG)、总胆固醇(TC)采用酶法测定。高密度脂蛋白胆固醇(HDL)、低密度脂蛋白胆固醇(LDL)采用均相酶法测定。脂蛋白a[Lp(a)]、载脂蛋白A1(ApoA1)、载脂蛋白A2(ApoA2)、载脂蛋白B(ApoB)、载脂蛋白C2(ApoC2)、载脂蛋白C3(ApoC3)、载脂蛋白E(ApoE)采用免疫透射比浊法测定。超敏C反应蛋白(hsCRP)采用颗粒增强的免疫散射比浊法测定。白细胞介素-6(IL-6)采用细胞因子芯片检测。通过实时荧光PCR方法、直接DNA测序、聚合酶链反应-限制性片段长度多态性(RFLP)三种检测方法的比较，分析其对77例样本检测结果的一致性。结果一、在国内首次成功建立了同时检测IL-6基因启动子区-572G／C、-597G／A多态性的实时荧光PCR检测方法。用DNA测序方法验证检测结果正确。经对中国北方地区汉族123例健康对照和194例冠心病患者的IL-6基因检测，发现在-597位点仅有两种基因型，为GG型和GA型，尚未发现AA型。-572位点有三种基因型，分别为GG，GC，CC型。其中-597GA型为国内首次报道。二、实时荧光PCR方法、直接DNA测序、RFLP三种检测方法的比较分析发现：在77例样本中，采用实时荧光PCR方法与直接DNA测序相比仅有3例结果不一致。77例PCR产物用限制性内切酶BsrBI酶切，有20例与实时荧光PCR方法结果不一致。当PCR产物用限制性内切酶FokI酶切时，仅有5例是GA型，另外两例未被检测出。三、统计分析发现IL-6基因启动子区-572G／C基因型频率和等位基因频率在冠心病组和对照组中的分布差异无统计学意义。携带G等位基因(GG+GC)和非G等位基因(CC)频率在冠心病组和对照组中的差异有统计学意义。在正常对照组中，与非G等位基因相比，携带G等位基因组的收缩压中位数水平显著升高。在所有观测对象中，与非G等位基因相比，携带G等位基因组的BMI、hsCRP、收缩压中位数水平显著升高。四、Logistic回归分析显示，ApoC2、TH、lip(a)、年龄、TG、性别、高血压是本研究对象冠心病发生的独立危险因素，ApoAl是保护因子，未见G等位基因是一个独立的危险因子。结论实时荧光PCR方法在30分钟内能够完成32例样本两个位点的同时检测。与DNA测序、RFLP方法相比，具有简单、准确、快速的特点，便于临床实验室大规模样本筛查。本研究未发现IL-6基因启动子区-597G／A多态性与CHD的易感性有关，但发现-572G／C多态性与CHD的易感性有关。其机制可能与-572G／C多态性可导致BMI、hsCRP和血压的变化有关。更多还原

【Abstract】 ObjectiveIn order to explore the associations between IL-6 gene polymorphisms and CHD, a rapid genotyping assay for the IL-6 gene by fluorescent resonance energy transfer (FRET) and melting curves was developed. The association between polymorphisms and CHD was explored by investigating the polymorphisms (-572G/C, -597G/A) in IL-6 gene promoter area and body mass index (BMI), inflammatory factors and other biochemistry parameters in Han nationality of North China. MethodsWe described a real-time polymerase chain reaction method for detecting the -572G/C and -597G/A promoter polymorphisms of IL-6 gene in Han nationality of North China. Serum TC and TG levels were determined with standard enzymic colorimetric methods. Serum HDL and LDL levels were determined with homogeneous enzymatic colorimetric assay. Concetrations of Lp (a), ApoA1, ApoA2, ApoB, ApoC2, ApoE and ApoC3 were measured by immuno-turbidirnetric assay (ITA). Serum hsCRP were determined by particle enhanced immunonephelometry. Serum IL-6 levels were detected by Cytokine panel. The concordance of 77 samples were analysed by real-time PCR, DNA sequence analysis and restriction fragment length polymorphism (RFLP) assay. ResultsA real-time PCR method was developed successfully and DNA sequencing analysis validated the results. 190 CHD patients and 123 controls were analysed. The data are the first time to show that genetype of IL-6 gene promoter -597G/A polymorphism in 7 cases were GA and were GG in others, whereas no AAgenotype had been found. GG, GC, and CC genotypes were also found of IL-6 gene -572G/C polymorphisms.Compated real-time PCR method with DNA sequence analysis, we found that only three samples were different in 77 samples. However, when the PCR product of 77 samples was digested by restriction enzyme BsrBI, 20 of them were different from real-time PCR method. The PCR product was digested by restriction enzyme Fok I, only five cases were found to be the GA genotype.Statistc analysis shows that no difference had been found between the frequencies of IL-6 gene -572G/C genotypes and alleles in CHD and control group. Significant difference was found between the G allele carrier (GG+GC) and non-G allele carrier (CC) in CHD group and control group. In the control group, median levels of systolic blood pressure of G allele carriers were significantly higher than that of non-G allele carriers. Among all the subjects, BMI, hsCRP and systolic blood pressure in the group of G allele carrier were significant higher than the group of non-G allele carrier.Multivariate logistic regression analysis showed that ApoC2, TH, lip (a), age, TG, sex and high blood pressure were risk factors for CHD. ApoAl was a protective factor. G allele was not found to be an independent risk factor. ConclusionTwo mutation sites of 32 samples could be detected simultaneously within 30 min. Compared with RFLP analysis and DNA sequencing analysis, it is a simple, accurate, and fast method, which is more suitable for large-scaled screen in clinical laboratory. The results indicated that at the two mutation sites, only the IL-6 gene -572G/C polymorphism might be correlated with susceptibility to CHD in Han nationality of North China. The mechanism may be related with the changes of BMI, hsCKP and blood pressure levels resulted from the polymorphism IL-6 -572G/C.更多还原