应用基因微阵列技术筛选法乐四联症相关基因

Screening of Tetralogy of Fallot-Associated Genes Using Gene Microarrays

目的:观察法乐四联症(TOF)相关基因的表达变化。方法:使用Biostar 80s型人类基因表达谱微阵列初步筛选TOF和室间隔缺损患者右心室心肌的差异表达基因。结合初筛实验、网上孟德尔人类遗传数据库及心脏基因表达数据库的查询结果,遴选补充至500条目标基因。应用3张定制的1000点人类基因微阵列,双重点样,分别筛选TOF(组)25例、不伴有先天心脏畸形的引产胎儿(FETAL组)5例、右室双出口(组)5例、室间隔缺损(组)15例患者,检测右心室流出道肌肉中的差异表达基因,进行生物信息学分析。采用实时荧光定量聚合酶链反应技术验证微阵列结果。结果:用Biostar 80s微阵列发现186条差异表达基因,用定制微阵列则分别发现104条(TOF/FETAL)、13条(TOF/右室双出口)和315条(TOF/室间隔缺损)。剔除结果可能不可靠者(179条),根据差异表达特征,将剩余基因归入14个相关基因群。实时荧光定量聚合酶链反应证实,胰岛素样生长因子结合蛋白3、Ⅰ型胶原α_2链蛋白与Ⅲ型胶原α_1链蛋白3个基因的差异表达与微阵列结果相一致。结论:应用基因微阵列技术进行高通量并行分析,可确定与TOF相关的基因表达谱。胶原家族成员及胰岛素样生长因子结合蛋白3可能与TOF的发生发展过程密切相关。

Objective： To screen the expression profiles of Tetralogy of Fallot （TOF）-associated genes. Methods ： A human high-density eDNA microarray （ Biostar80s ） was used to examine the differentially expressed genes in right ventricnlar myocardium with TOF or ventricnlar septal defect （VSD）. Based on the data of Biostar 80s microarray and the information from the OMIM （online mendelian inheritance in man） and CaGE （Cardiac Genes Expression Knowledgebase） databases, 500 candicate genes were selected for this study. Then, the bio-informatical analysis on the gene expression differences among the right ventricular myocardium of patients with TOF, VSD, double outlet right ventricle （ DORV ）, and normal fetus were carried out by means of three human cDNA microarrays, which were designed and prepared with 1000 hybridization points. Three of the ditterentially expressed genes were further confirmed using real-time PCR. Results： 186, 104, 13,315 genes/ESTs were found to be differentially expressed in Biostar 80s （TOF/VSD） , Chipl （TOF/ FETAL）, chip2（ TOF/DORV）, and chip3 （TOF/VSD） ,respectively. Finally 179 genes which are not believed to be related to TOF were excluded and the remaining genes were divided into 14 gene clusters according to the characteristics of differentially expressed genes. Real-time PCR was carried out to confirm the results of differentially expressed genes of insulin-like growth factor binding protein 3（ IGFBP3 ） , COL1A2 and COL3A1 in microarrays. Conclusions：Microarray gene expression profile analysis is demonstrated to be an advanced technique for high-throughput screening of the related genes of TOF. Studies of the expression profile for genes of interest, which may involved in the genesis and development of TOF, are helpful for the understanding of its pathogenesis and pathophysiology. Changed expression of the members of collagen family and IGFBP3 were assumed to be associated with the development of TOF.