应用抑制性消减杂交筛选失血性休克所致大鼠心肌差异表达基因

Screening of differentially expressed genes in rat hearts after hemorrhagic shock with suppression subtractive hybridization

目的 失血性休克可导致大鼠心肌的缺血、缺氧 ,进而对心肌细胞造成严重损伤 ,本实验将利用抑制性消减杂交技术(Suppressionsubtractivehybridization ,SSH) ,研究失血性休克大鼠心肌的差异表达基因 ,以期通过基因线索寻找其损伤机制。方法 建立失血性休克大鼠模型 ,选其心肌 ,提取总RNA ,构建cDNA文库 ,应用抑制性消减杂交技术筛选其差异表达基因。通过反向North ern杂交验证差异表达基因阳性克隆 ,并进行测序分析 ,登陆Genbank寻找同源性基因。结果 获得 42个阳性结果 ,2 5个为高表达基因 ,17个为低表达基因 ,其中发现 5个新的cDNA片段。结论 SSH技术是一种高效的筛选差异基因的方法 ,本实验发现失血性休克可导致大鼠 45SrDNA基因转录起始区域、鼠半胱氨酸富含蛋白 6 1及鼠血清诱导激酶等基因的差异表达 。

Objective To investigate genetic clues to reveal the mechanisms of severe heart injuries caused by hemorrhagic shock, especially by hypoxia and ischemia in rats. Methods Hemorrhagic shock rat models were established and total RNA were extracted from rat hearts. After the synthesis of the dscDNA, differentially expressed genes was isolated with suppression subtractive hybridization (SSH). Then the isolated genes were cloned and sequenced. The sequencing results were compared with their counterparts in Genbank to search for their homology. Results Among the 42 obtained positive genes, 25 were highly expressed and 17 lowly. Additionally, 5 new sequences were found. Conclusion SSH is an effective method to isolate differentially expressed genes. Hemorrhagic shock changes the expressions of some genes in rat heart, such as Rat 45S rDNA gene transcription initiation region, Rattus norvegicus cysteine rich protein 61 and Rattus norvegicus serum inducible kinase (Snk). But the relation between these genes and the injuries needs further study.