【作者】 袁灿；

【导师】 肖献忠；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2003， 博士

【摘要】 短暂的心肌缺血可同时导致两种不同的后果—心肌顿抑和缺血预适应。这两种现象的存在表明,当受到缺血损伤刺激时,心肌可调动自身的内源性保护系统,适应和抵抗损伤。揭示这种内源性保护现象的分子机制,对缺血性心脏病的防治具有重要意义。短暂的心肌缺血-再灌注可以诱导许多具有细胞保护作用的基因表达上调,这些表达上调的基因是心肌内源性保护分子机制的重要组成部分。本文采用大鼠心肌短暂缺血-再灌注动物模型,运用抑制消减杂交方法,构建了大鼠心肌短暂缺血-再灌注诱导表达上调基因的消减cDNA文库。通过反向Northern点杂交从文库中筛选了85个克隆进行测序。通过与GenBank数据库进行序列比对,获得了31个已知基因和18个可能代表新基因的EST片段。从以上所获的差异表达基因中任选5个进行RT-PCR分析,任选两个进行Northern杂交分析,均证实其在缺血-再灌注组心肌组织中表达上调。在所筛到的31个已知基因中,多个基因已有文献报道在心肌缺血-再灌注时表达上调并发挥心肌保护作用,另一些已知基因与心肌缺血-再灌注的关系至今未见报道。筛选得到的18个可能代表新基因的EST片段已在GenBank中登录。对以上已知基因进行进一步功能分析和对未知基因进行克隆,可能为揭示心肌的内源性保护分子机制提供新的线索和思路。心肌缺血-再灌注后的损伤修复及适应代偿是一个动态变化过程,并涉及复杂的基因调控网络。为了从基因组水平反应这种动态变化过程并探讨其相关机制,我们采用含4097个基因点的大鼠cDNA芯片分析了大鼠心肌短暂缺血-再灌注后不同时间点(1、3、6、12、24小时)的基因表达谱情况。结果显示,再灌3小时表达改变的基因数最多,而再灌1小时表达改变的基因数最少。选择至少在两个时间点以上表达发生改变的基因,采用自组织图算法将具有相同或相似表达模式的基因聚为12类。基因表达的各种模式提示,不同的基因参与调控心肌缺血-再灌注后不同时间点的功能代谢变化过程,且聚为一类的基因可能具有相似的表达调控机制。以上结果为进一步深入研究心肌缺血-再灌注后功能代谢变化的分子机制及其复杂基因调控网络提供了线索和思路。此外,筛选到的一些可能代表新基因的EST片段也将为克隆新的心肌缺血-再灌注反应相关基因提供线索。从以上方法所获得的EST片段中,我们采用生物信息学方法和5更多还原

【Abstract】 Brief episodes of myocardial ischemia may result in two phenomena: myocardial stunning and preconditioning. Clarifying the mechanisms of myocardial stunning and preconditioning could improve the prevention and treatment of ischemic heart diseases.A number of genes have been shown up-regulated during myocardial ischemia/reperfusion and playing important roles in myocardial protection, but there are clearly many missing elements to be found. We used suppression subtractive hybridization to construct cDNA libraries enriched for genes up-regulated during transient ischemia/ reperfusion of the rat’s hearts. After being confirmed by reverse northern dot blot for differential expression, 85 selected genes were sequenced and searched in Genbank for homology analysis. The differential expression of the genes was further confirmed by RT-PCR and Northern blot analysis. Of 85 selected genes, 31 genes were nuclear- encoded genes, while some of them had been found up-regulated during ischemic preconditioning and participated in cytoprotection in previous literature, others were never found related to myocardial ischemia/reperfusion injury before this study. 18 novel ESTs which may represent new genes have been registered into Genbank with accession numbers .We further used cDNA microarray to analyze myocardial gene expression profile in rats during myocardial ischemia/reperfusion at multiple time points ( 1, 3, 6, 12, 24 hours after reperfusion). Gene expression peaked at 3 hours after reperfusion was lowest at 1 hour after reperfusion. Using self-organizing map (SOM) algorithm, we clustered the genes which changed in expression at two or more time points after reperfusion into twelve clusters and each cluster showed a similar expression pattern. Analysis of gene expression patterns revealed sequential induction of subsets of genes after transient myocardial ischemia/reperfusion that characterize each response. Genes in the same cluster might be regulated in a similar manner.Using bioinformatics analysis and 5′RACE technology, we cloned a new gene Mip1 which was up-regulated after transient更多还原