DEPP蛋白对血管内皮细胞的存活及基因表达的影响

Effect on cell viability and gene expression in human umbilical endothelial cell EA.hy926 by adenovirus mediated DEPP over-expression

目的:探讨孕酮诱导的蜕膜蛋白(decidual protein induced by progesterone,DEPP)在血管内皮细胞EA.hy926中的定位、对细胞存活及基因表达的影响。方法:构建表达人DEPP的重组腺病毒,测定病毒滴度并鉴定其表达的DEPP蛋白水平。用构建好的腺病毒感染EA.hy926内皮细胞,通过免疫荧光和Western blot确定DEPP蛋白在细胞中的定位。观察DEPP过表达对EA.hy926细胞存活的影响,用MTT法测定细胞的存活率,通过流式细胞仪检测EA.hy926细胞的坏死和凋亡情况。利用蛋白质芯片检测DEPP过表达时内皮细胞中血管发生相关基因表达水平的变化。结果:本研究成功构建了表达人DEPP蛋白的重组腺病毒。通过免疫荧光检测,发现DEPP蛋白呈全细胞分布。与过表达GFP的对照细胞相比,过表达DEPP时EA.hy926细胞明显皱缩、变圆,MTT检测显示细胞的存活率明显下降,流式细胞仪检测显示EA.hy926细胞感染腺病毒后细胞的坏死水平与对照组相比升高。蛋白质芯片检测发现,DEPP过表达导致内皮细胞中多种血管发生相关蛋白的水平发生明显改变。结论:DEPP蛋白的表达升高对血管内皮细胞有损伤作用,可能参与血管发生的调控过程。

Objective：To investigate the subcellular localization of decidual protein induced by progesterone （DEPP） as well as its effect on cell viability and gene expression in human umbilical endothelial cell line EA.hy926. Methods： Recombinant adenovirus expressing human DEPP was generated. The snbcellular localization of DEPP was investigated by using immunofluorescence and Western blot assay with cells infected by ADV-DEPP. Cell viability was determined by using MTT assay. Apoptotic or necrotic cell numbers were measured by flow cytometry. Angiogenesis related protein expression pattern in response to DEPP over-expression was examined by using antibody microarray, Results：Over-expressed DEPP was localized both in the nucleus and cytoplasm in EA.hy926 cell. Compared with control cells,DEPP expression led to more severe cell shrinkage and detachment after adenovirus infection. MTF assay showed that cell viability was significantly decreased with exogenous DEPP. Flow cytometry analysis revealed that the main cell death induced by DEPP was necrosis. Finally,angiogenesis antibody microarray indicated that DEPP induced the increase of 7 genes and the reduction of 70 genes expression in endothelial ceils. Conclusion：With increased expression, DEPP led to endothelial cell damage and may be involved in the regulation of angiogenesis.