应用抑制消减杂交克隆内皮细胞内毒素刺激后相关基因  

CLONING OF LIPOPOLYSACCHARIDE RESPONSIVE GENES IN ENDOTHELIAL CELLS BY SUPPRESSION SUBTRACTIVE HYBRIDIZATION

作　　者：梁自文[1] 罗向东[1] 杨宗城[1] 

机构地区：[1]第三军医大学西南医院

出　　处：《解放军医学杂志》2002年第4期336-338,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金重点资助项目 (编号 39830 4 0 0 )

摘　　要：提取人脐静脉内皮细胞经内毒素刺激 6h后的mRNA并合成cDNA ,与对照组进行抑制消减杂交 ,经菌落斑点杂交筛选出阳性克隆 ,进行测序和同源性分析 ,并采用Northern杂交验证新的cDNA序列。共获得 2 5条差异表达基因 ,涉及与编码炎症介质、胞内信号传导、细胞骨架、细胞凋亡和能量代谢相关的基因 ,其中 3条为新基因序列。结果表明 ,抑制消减杂交技术有效地克隆了内皮细胞内毒素刺激后相关基因 ,有助于阐明内毒素激活血管内皮细胞的机制 。To screen genes in endothelial cells resulted from responding to lipopolysaccharide (LPS), mRNA was extracted from both untreated human umbilical endothelial cells (HUVEC) following and HUVEC which were treated with LPS for 6 hours. cDNAs of both populations were synthesized to generate cDNA libraries by suppression subtractive hybridization (SSH). The libraries were then screened with colony dot blots. Positive clones were sequenced and BLAST analysed. The results showed differential genes included 3 novel genes and 22 known genes. The 3 novel genes were confirmed by Northern blotting analysis. These 22 known genes were involved in the regulation of proinflammatory response, cell apoptosis, cytoskeleton, signal transduction and energy metabolism. These results suggest that SSH is an effective technique to detect differential gene expression in HUVEC, which may be helpful to evaluate molecular mechanisms of endothelial injury induced by LPS and provide potential therapeutic targets for LPS related disturbances.

关 键 词：抑制消减杂交 克隆 内皮细胞 内毒素 相关基因 分子机制 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]