A20基因抑制内皮细胞组织因子表达的研究

A20 gene inhibits LPS-induced tissue factor expression of endothelial cells

目的 探讨外源性A2 0基因在内皮细胞获得表达及对脂多糖诱导的内皮细胞组织因子表达的影响。方法 DOTAP脂质体介导的pCAGGSEHA2 0和pMAMneo基因转染 ,经G4 18筛选阳性单克隆 ,再用免疫荧光组织化学检测A2 0基因的表达 ,将转染A2 0基因的内皮细胞与未转染的内皮细胞分别加脂多糖 ,经原位杂交及免疫荧光组织化学检测组织因子基因的转录和表达 ,采用裂解内皮细胞一步复钙凝块时间测定法测定组织因子促凝活性。结果 成功构建了pCAGGSEHA2 0真核表达重组体 ,A2 0基因在经G4 18筛选后的内皮细胞中得到有效表达 ,能抑制 70 %组织因子的表达及凝块的形成。结论 A2 0基因能够明显抑制脂多糖诱导的内皮细胞组织因子的表达 。

Objective To examine the effect of exogenous human A20 gene transferred into endothelial cells on LPS induced tissue factor(TF) expression of endothelial cells.Methods pCAGGSEHA20 was constructed using gene cloning and recombination technique.With the help of DOTAP,endothelial cells were transfected with pCAGGSEHA20 and pMAMneo.The postive cell clones were selected with G418.The stable transfection and expression of A20 gene in the endothelial cells were determined by immunofluorescence analysis.Lipopolysaccharide(LPS) was added to the transfected cells and untransfected cells,and expression of TF in the endothelial cells was determined by in situ hybridization and immunofluorescence analysis.TF activity was determined by one step recalcification clotting time assay.Results The construction of pCAGGSEHA20 was successful.Abundant A20 gene stable expression in endothelial cells transfected with pCAGGSEHA20 was confirmed by immunofluorescence analysis.A20 gene could inhibit 70% LPS induced TF expression and reduce 76.4% of LPS induced TF activity in endothelial cells.Conclusion With the help of DOTAP,hA20 gene can be transferred and stably expressed in endothelial cells and it can inhibit LPS induced TF expression in endothelial cells.