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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 ,并为临床防治内毒素损害寻找新的作用靶点提供了根据
The mRNA of human umbilical vein endothelial cells (HUVECs) stimulated by endotoxin for 6h was extracted and cDNA was synthesized. The cDNAs were screened by suppression subtractive hybridization with the control group. The positive clones were screened by dot blot hybridization. Sequencing and homology analysis were performed. Northern hybridization cDNA sequence. A total of 25 differentially expressed genes were obtained, involving genes encoding inflammatory mediators, intracellular signaling, cytoskeleton, apoptosis and energy metabolism, of which 3 were novel gene sequences. The results showed that the suppression subtractive hybridization technique effectively cloned endothelial-related genes after endotoxin-induced stimulation, which may be helpful to clarify the mechanism of endotoxin-activated vascular endothelial cells and provide a basis for finding a new target for the prevention and treatment of endotoxin damage