低压缺氧延迟预适应小鼠海马差异表达基因的筛选及鉴定研究

Screening and identification of differentially expressed genes in hippocampus of mice during hypobaric hypoxic delayed preconditioning

目的:探索低压缺氧延迟预适应小鼠海马差异表达基因。方法:将近交系Babl/c小鼠放入减压舱,模拟海拔7000m高度减压2·5h/d,连续3d。第3次减压毕36h后,提取海马总RNA,SMARTPCR合成cDNA,经抑制消减杂交,建立消减cDNA文库。随机挑取文库单克隆,用反向Northern杂交,分别对来自于正、反向文库的452个和74个克隆进行了筛选。结果:用消减探针筛选时,217个基因片段的杂交信号在延迟预适应海马增加2倍以上,85个基因片段的杂交信号则降低66%以下。用未消减探针筛选时,135个基因片段的杂交信号在延迟预适应海马增加2倍以上,44个基因片段的杂交信号则降低66%以下。对部分基因片段测序显示,小鼠线粒体细胞色素C氧化酶亚单位1、线粒体NADH脱氢酶亚单位1和6、小鼠DSS1和克隆号为IMAGE:3582855的基因在延迟预适应海马表达增加。克隆号为IMAGE:3593193的基因、与成年雄性小鼠嗅脑的一个cDNA高度相似的基因及与小鼠bladder RCB-0544MBT-2cDNA高度相似的基因在延迟预适应海马表达降低。结论:小鼠海马低压缺氧延迟预适应多种基因表达发生改变,它们可能是产生低压缺氧延迟预适应的重要机制之一。

AIM： This study was designed to explore the differentially expressed genes between hypobaric hypoxic delayed preconditioning （HHDP） and normal mouse hippocampus. METHODS： HHDP was produced by treating the animals at a 7 000 m high altitude for 2.5 h/d for 3 d. At 36 h after last time decompression, total RNA was isolated from hippocampus, cDNA was synthesized and amplified by SMART PCR. cDNA libraries of differentially expressed gene between HHDP and control hippocampus were constructed. 452 clones from forward （ subtracted control from preconditioning） cDNA library and 74 clones from backward （subtracted preconditioning from control） one were screened by reverse Northern hybridization. RESULTS： Screening with subtracted probes, hybridization signal of 85 gene fractions decreased and that of 217 gene fractions increased by more than 2 times in HHDP hippocampus compared with control. Screening with unsubtracted probe, hybridization signal of 44 gene fractions decreased and that of 135 gene fractions increased by more than 2 times in HHDP hippocampus compared with control. Some of the clones had been sequenced. Analysis and comparison with the data of GenBank were performed. The results showed that mouse cytochrome C oxidase subtmit 1, NADH dehydrogenase subunit 1 and 6, deleted in split - hand/split - foot 1 region （ DSS1 ） and cDNA corresponding to clone IMAGE： 5251089 of mice cDNA library were increased in hippocampus of HHDP mice. cDNA correspending to clone IMAGE： 3593193, mus musculus adult male olfactory brain cDNA and mus musculus bladder RCB - 0544 MBT - 2 cDAN were decreased in hippocampus of HHDP mice. CONCLUSION ： Many genes expresses differentially in hippocampus of mice during HHDP. This may be one of the molecular mechanisms of HHDP.