人脐静脉内皮细胞脂多糖刺激后上调基因表达谱的分析

Analysis of up-regulated genes in human umbilical vein endothelial cells stimulated by lipopolysaccharide

目的 对人脐静脉内皮细胞 (HUVEC)受脂多糖 (LPS)刺激后上调基因表达谱进行筛选及分析。方法 以受LPS刺激HUVCE为实验方 ,未刺激HUVEC为驱使方 ,进行抑制消减杂交 (SSH)建立消减cDNA文库 ,经菌落原位斑点杂交筛选文库后将阳性克隆测序和同源性分析 ,并用RT PCR验证新的cDNA序列。结果 共获得 2 5条上调表达的基因 ,分别与炎症反应、细胞骨架重构、细胞生长和凋亡、胞内信号转导相关 ,并克隆到 3条新基因表达序列标签 (EST) ,RT PCR验证了这些新EST序列只在LPS刺激的内皮细胞中表达。结论 抑制消减杂交有效地克隆了人脐静脉内皮细胞LPS刺激后上调表达基因 。

Objective To screen and analyze genes up regulated in human umbilical vein endothelial cells (HUVEC) stimulated by lipopolysaccharide (LPS). Methods Suppression subtractive hybridization (SSH) was performed between the unstimulated HUVEC(driver) and HUVEC stimulated with LPS(tester) to generate subtractive cDNA library. The library was screened with colony dot hybridization to further verify the differentially expressed cDNA clones. Positive clones were sequenced and BLAST analyzed. The 3 novel cDNA sequences were verified by RT PCR. Results Twenty five up regulated genes related to inflammation, cellular cytoskeletal rearrangement, cellular proliferation and apoptosis, intercellular message transduction, and 3 new expression sequence tags (EST) were acquired. RT PCR indicated the expression of the new ESTs only in HUVEC stimulated by LPS. Conclusion SSH is a powerful technique of high sensitivity for the detection and clone of up regulated gene expressed in HUVEC stimulated by LPS, which may be helpful to clarify the mechanism of endothelial cells activation stimulated by LPS.