应用抑制消减杂交技术筛选颞叶癫痫的相关基因

Application of suppression subtractive hybridization to screen for temporal lobe epilepsy-related genes

目的构建颞叶癫痫大鼠海马组织差异表达基因的消减cDNA文库,筛选颞叶癫痫的差异基因。方法采用抑制消减杂交方法,以正常大鼠和癫痫大鼠的海马组织作为对比材料,分离组织中差异表达基因的cDNA片段,将其与T载体进行T/A连接构建文库,将连接产物转化大肠杆菌进行文库扩增后,随机挑取100个白色克隆,用菌落PCR法筛选差异基因克隆,并进行测序分析。结果获得14个在癫痫大鼠海马组织中有差异表达,并与大鼠的已知基因片段高度同源的基因。结论抑制消减杂交技术是一种高效的筛选差异表达基因的方法,颞叶癫痫大鼠海马组织中存在许多差异表达基因,为深入研究癫痫发病的分子机制提供重要线索。

Objective To construct a subtracted cDNA library of differentially expressed genes in the hippocampus of rats with temporal lobe epilepsy,and to screen for differentially expressed genes related to temporal lobe epilepsy.Methods Suppression subtractive hybridization（SSH） was used for isolating cDNA fragments of differentially expressed genes in the hippocampus of temporal lobe epilepti rats,and normal hippocampus tissues were controls.The products of SSH were directly inserted into the T/A cloning vector to set up the subtractive library which was amplified through E.coli transformation.One hundred positive bacterium clones were randomly picked and screened the colony polymerase chain reaction（PCR） method.Results Fourteen differentially expressed genes were obtained from the hippocampus of temporal lobe epileptic rats by sequencing and using Basic Local Alignment Search Tool（BLAST）,and they were highly homologous with the known gene fragments in rats.Conclusion SSH is an effective method for screening for differentially expressed genes.Many differentially expressed genes exist in the hippocampus of rats with temporal lobe epilepsy.The results of this study provide an important clue for further exploring the molecular mechanism of temporal lobe epilepsy.