A2 0基因在血管内皮细胞的表达研究

TRANSFECTION OF ENDOTHELIAL CELLS WITH pCAGGSEHA20 AND ITS STABLE EXPRESSION

目的 探讨外源性A2 0基因在内皮细胞获得稳定表达的可行性。 方法 将N 末端标记E tag的HA2 0cDNA重组于pCAGGS真核表达载体 ,构建了pCAGGSEHA2 0真核表达重组体。DOTAP脂质体介导的pCAGGSEHA2 0和pMAMneo基因转染 ,经G4 18筛选阳性克隆 ,再用免疫荧光及分子原位杂交检测A2 0基因的表达。 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。 结论 在DOTAP脂质体介导下 ,A2

Objective To observe the effectiveness of transferring human A20 gene into endothelial cells. Methods The shuttle plasmid pCAGGSEHA20 was constructed using gene cloning and recombined technique. Endothelial cells were transfected with pCAGGSEHA20 and pMAMneo by DOTAP. The postive cell clones were selected with G418. The stable transfection and expression of A20 in the endothelial cells were determined by in situ hybridization and immunohistochemical analysis. Results The two fragments digested from pCAGGSEHA20 by EcoRⅠ represented 4 6?kb and 2 3?kb by electrophoresis, which were confirmed to be the carrier and the A20 gene fragments inserted originally. The above results indicate that the construction of pCAGGSEHA20 was successful. Abundant A20 stable expression in endothelial cells transfected with pCAGGSEHA20 was confirmed by in situ hybridization and immunohistochemical analysis.Conclusion By means of the DOTAP, hA20 gene can be transferred and stably expressed in endothelial cells.