【作者】 张阳东；

【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， 2009， 博士

【摘要】 [目的]近年来我国CAD的患病率和死亡率呈逐年上升趋势。CAD是遗传因素和环境因素共同作用导致粥样硬化斑块在冠状动脉壁沉积而引起的。为了寻找与CAD相关的SNP位点和基因,本课题构建了多重SNP基因分型和多重基因表达检测方法,并对CAD患者和健康对照人群外周血白细胞的多个SNP位点和多个目标基因的表达水平进行了检测。[方法](1)多重SNP基因分型的检测:提取外周血白细胞DNA,选择HapMap上公布的ADD1,PECAM-1,CRP,ecNOS,PC-1,SELL,GNB3,ACE,AT1R,AGT,ICAM-1,MTHFR和HL基因中国汉族人群的44个标签SNP,每个SNP设计3条引物(2条PCR扩增引物和1条延伸引物),通过PCR扩增、延伸及杂交,应用SNP stream基因分型系统对SNP位点进行基因分型。(2)多重基因表达水平的检测:提取外周血白细胞RNA,选择MTHFR,IL-8,ecNOS,TLR4,FOS,ID2,ADD1,ICAM-1,PECAM-1,CRP,LDLR,vWF,SELL,TNF-α等14个目标基因和β-actin、SRP14及18S-rRNA等3个管家基因,每个基因设计2条引物(1条反转录引物和1条PCR扩增引物),通过反转录和PCR扩增,应用高通量的GenomeLab GeXP分析仪检测17个基因的表达水平。(3)应用全自动生化分析仪检测血脂、CRP、ACE及Hcy水平。[结果](1)构建的44个SNP分型成功32个,分型成功率为72.7%(32/44)。通过对583份DNA标本进行检测,32个SNP的平均样本检出率为98.3%。通过对中国汉族CAD患者和健康对照人群的32个SNP进行检测,结果显示CRP,ecNOS,tPc-1和ACE基因中的6个SNP的基因型频率和(或)等位基因频率在两组间存在统计学差异。根据连锁不平衡水平构建单倍型,结果显示ecNOS,PC-1,ACE基因的单倍型频率与CAD相关。实验发现CRP,ACE和MTHFR基因多态性分别与血清CRP,ACE和Hcy水平相关。(2)通过单重基因扩增、电泳,确认每个基因扩增产物的大小。结果显示扩增产物的大小与设计大小基本一致。根据每个基因扩增产物、质控基因和内标的片段大小,确认实验设计的每个基因扩增产物的特异性。在多重基因扩增中,根据电泳结果对外周血白细胞中高丰度表达基因的引物进行倍比稀释,确定了体系中每个基因的最佳引物浓度。通过对3个管家基因在两组之间表达水平的比较,发现以β-actin和SRP14基因的几何均数作为标准化因子可用以标准化本体系目标基因的表达。应用上述构建的14重基因表达检测方法对CAD患者和健康对照人群外周血白细胞RNA的基因表达水平进行检测,发现ecNOS,SELL,ADD1和MTHFR基因的表达水平在两组间存在统计学差异,其表达水平与部分SNP的基因型有关。[结论]本实验构建的32重sNP基因分型和14重基因表达检测方法具有快速、准确、高通量、操作简单、微量化等优点。实验发现的与CAD相关的6个sNP位点和4个表达差异基因为心血管危险因素的预测和诊断提供了一种新思路,为心血管疾病寻找更有效的防治途径,早期预防和及时治疗提供理论指导。更多还原

【Abstract】 [Objective]Coronary artery disease(CAD) is the leading cause of morbidity and mortality in recent years in China.It is a multifactorial disease caused by various genetic and environmental factors involved in accumulation of atherosclerosis in the walls of the coronary arteries.In order to find single nucleotide polymorphisms(SNP) and genes that were associated with CAD, multiplexed SNPs genotyping and genes expression methods were established in the current work.With the multiplexed methods,lots of tag SNPs and genes in peripheral blood white cells from CAD patients and normal control individuals were investigated.[Methods](1) The method of multiplexed SNPs genotyping:DNA samples were extracted from peripheral blood white cells and 44 tag SNPs of 13 genes (ADD1,PECAM-1,CRP,ecNOS,PC-1,SELL,GNB3,ACE,AT1R,AGT, ICAM-1,MTHFR and HL) that were listed in the International Haplotype Map Project Database for CHB(Chinese Han nationality in Beijing) were selected in the current work.Three primers were designed for each SNP(two for polymerase chain reaction(PCR) and one for extension reaction).After PCR, extension and hybridization,genotypes of SNPs were investigated with the GenomeLab SNPstream genotyping system.(2) The method of multiplexed genes expression:RNA samples were extracted from peripheral blood white cells and two primers(one for reverse transcription and one for PCR) were designed for each of the 14 target genes(MTHFR,IL-8,ecNOS,TLR4,FOS, ID2,ADD1,ICAM-1,PECAM-1,CRP,LDLR,vWF,SELL and TNF-α) and 3 house keeping genes(β-actin,SRP14 and 18S-rRNA).After reverse transcription and PCR,expression levels of the 17 genes were detected with high-throughput GenomeLab GeXP Genetic Analysis System.(3) The levels of serum lipid,CRP,ACE and Hcy were detected with automatic biochemical analysis system.[Result](1) Thirty two SNPs were genotyped and the rate of succeed SNP genotyping was 72.7%(32/44).The average genotyped rate of the 32 SNPs was 98.3%in the test of 583 DNA samples.With the 32 plexes SNPs genotyping method,DNA samples of CAD patients and normal control individuals from the local ethnic Han Chinese population were investigated.The results showed that the genotype and/or allele frequencies of 6 tag SNPs in CRP,ecNOS,PC-1 and ACE genes were significantly different in both groups.According the linkage-disequilibrium levels of SNPs,the haplotypes were constructed and the results showed that the frequencies of the constructed haplotypes of ecNOS, PC-1 and ACE genes were associated with CAD.Genotpyes of CRP,ACE and MTHFR genes were associated with serum CRP,ACE and Hcy level.(2) Confirmed by single plex PCR and capillary electrophoresis,the size of each gene’s PCR production was consistent with original designed size.The specific of each target gene was confirmed according the capillary electrophoresis size of each gene’s PCR production,control gene and size standard.The appropriate primer concentration of genes which were expressed abundant in peripheral blood white cells were confirmed by twofold serial dilutions in multiplexed PCR. In the current work we found the house keeping genes expression ofβ-actin and SRP14 were more stable than 18S-rRNA between the CAD and normal control groups,and the geometry average ofβ-actin and SRP14 could be regarded as normalization factor for quantification of target genes in the current work.We detected 14 genes expression profile in CAD patients and normal control subjects with the method described as above,and found expressions of ecNOS, SELL,ADD1 and MTHFR in the peripheral blood white cells were significantly different in both groups.The expression levels of these four genes were associated with genotypes of some SNPs.[Conclusion]Multiplexed SNPs genotyping and genes expression methods established in the current work were quick,high-throughput,easy operation and microanalyse.Six SNPs and 4 genes discovered in the work that were associated with CAD may help in explaining the molecular bases of the disorder and the susceptibility to coronary atherosclerosis.It also can guide early predicting, preventing and therapy for CAD.更多还原