室间隔缺损患者心肌组织相关基因的筛选及验证

【作者】 张浩；

【导师】 曹克将； 孔祥清；

【作者基本信息】 南京医科大学 ， 内科学， 2007， 博士

【摘要】 研究背景先天性心脏病(congenital heart disease，CHD)是人类最常见的出生缺陷之一，在活产婴儿中的发病率为0.6％～0.9％，而在自然流产婴儿中则高达5％～10％。室间隔缺损是先天性心脏病中最多见的类型，约占CHD发病的33％。目前认为，室间隔缺损的发病原因为环境和遗传两方面因素的共同作用结果。虽然现在可以采用外科手术或是介入手术对部分室间隔缺损进行修复治疗，但对其发生，发展等原因还尚未清楚。从遗传学角度，特别是基因水平上研究室间隔缺损的形成机制将为其早期预防提供良好的基础。应用基因差异表达分析技术，从转录水平筛选出室间隔缺损相关基因，结合生物信息学及分子生物学手段，进一步探讨筛选出室间隔缺损相关基因在其发生中的作用，为明确室间隔缺损的发病机制及其早期预防提供研究基础，甚至能够更加深入了解心脏的发育，生长，老化和疾病的机制。目的运用抑制性差减杂交技术构建双向差减cDNA文库，通过测序，生物信息学分析获得人类室间隔缺损与正常心脏的差异表达基因，为阐明室间隔缺损的病因机制建立实验基础。方法以室间隔缺损患者心肌组织作为检测组(Tester)，正常人心肌组织作为驱赶组(Driver)，进行正向SSH；反之，正常人心肌组织作为检测组(Tester)，室间隔缺损患者心肌组织作为驱赶组(Driver)，进行反向SSH。经过总RNA的提取，mRNA的纯化，反转录，酶切，接头连接，两轮差减杂交和两轮PCR，获得正向和反向两个cDNA文库，构建文库载体，并转化到感受态细胞中扩增，获得单克隆，进行测序及生物信息学分析，随机挑选部分差异表达基因，运用RT-PCR技术进行验证。结果(1)构建了两个分别含有648和730个重组子的正向和反向差减文库，插入片断大小约200～600bp；(2)通过测序获得1378个表达序列标签(expressed sequence tag，EST)序列，对其进行生物信息学分析，发现这些EST序列约对应于551个基因，其中299个基因在室间隔缺损心肌组织中高表达，252个则低表达。这些基因与能量代谢，细胞周期及生长发育，细胞骨架和细胞黏附，LIM蛋白，锌指结构蛋白和发育等密切相关；(3)经RT-PCR验证差减杂交结果证实，差减效率较高。结论RT-PCR验证部分基因的表达趋势与差减文库表达谱趋势一致，提示所构建文库的可靠性和可信度较高。随着对这些基因的进一步研究，能够更加深入认识室间隔缺损的病因机制，甚至对心脏发育，老化和疾病的认识提供一定的基础。更多还原

【Abstract】 Background Congenital heart defects (CHDs) account for the largestnumber of birth defects in human, with an incidence of 0.6%～0.9% inlive births and 5%～10% in spontaneously aborted pregnancies. As asingle cardiac malformation, the ventricular septal defect (VSD) is themost frequent form of CHDs and is present in the 33% of all affectedinfants.It is well established that environmental and genetic causes are bothcontributing to the development of VSD. Although the defect of VSD canbe repaired through surgery operation or trans-catheter intervention, thebasic mechanism of VSD is still unknown.Using SSH in the present study to identify differentially expressedgenes related to VSD could partly elucidate the mechanism of thedevelopment of VSD and lead to new aspect of diagnosis and primaryprevention of VSD, even the mechanism in cardiac development, aging,and disease.Objective Ventricular septal defect (VSD) accounts for the largestnumber of birth congenital heart defects in human, but the geneticprograms that control ventricular septation are poorly understood. UsingSSH in the present study to identify differentially expressed genes related to VSD could elucidate the mechanism of the development of VSD.Methods To identify differentially expressed genes between ventricularseptal defect and normal ventricular septum myocardium, we haveundertaken suppression subtractive hybridization (SSH) and generatedreciprocal cDNA collections of representative mRNAs specific to humanheart with ventricular septal defect versus normal control. The products ofSSH were inserted to vectors and transferred to competent Cells. Then,the clones were obtained after amplifying and sequenced, bioinfonnaticsanalysis later. Some randomly selected genes were further validated byRT-PCR.Results (1) Following SSH, forward and backward subtract librariesincluding 648 and 730 recons were built, and the length of insert partswas between 200bp and 600bp; (2) 1378 clones (expressed sequence tag,EST) were sequenced and found to derive from 551 different genes. Ofthese 551 unique genes, 299 genes were up-regulated and 252 genes weredown-regulated in VSD patient’s heart. These predominately expressedgenes included genes involved in energy metabolism, cell cycle andgrowth, cytoskeleton and cell adhesion, LIM protein, zinc finger proteinand development; (3) The validation of differential expression of genesby RT-PCR demonstrated the low false positive rate associated with SSHin this experiment.Conclusions The trends of validation of differential expression of genes by RT-PCR were consistent to the results of SSH, which suggestedthat the library was highly reliability and credibility. It is anticipated thatfurther study of genes identified will provide insights into their specificroles in the etiology of VSD, even in cardiac development, aging, anddisease.更多还原