人巨细胞病毒抑制SIRT1表达诱导内皮细胞血管新生的研究

摘要

研究背景 人巨细胞病毒(HCMV)与动脉粥样硬化、移植性血管硬化和动脉再狭窄密切相关.已有研究证实人巨细胞病毒感染可诱发血管新生继而引起血管性疾病.因此,本文拟进一步探讨人巨细胞病毒诱导内皮细胞血管新生的分子学机制.方法 分别于感染HCMV 2、6、12 和24 h 后收集血管内皮细胞EA.hy926,实时定量聚合酶链反应和Western blotting 法检测SIRT1 mRNA 及其蛋白质表达水平;通过激动剂Resveratrol或抑制剂SIRT1 siRNA 孵育内皮细胞,观察病毒感染24 h后其增殖、迁移和小管生成能力.结果 与模拟感染组相比,HCMV 感染组EA.hy926 细胞SIRT1 mRNA 表达无变化(F = 1.395,P = 0.304);而SIRT1 蛋白表达水平呈逐渐递减趋势(F = 23.927,P = 0.000).激动或抑制SIRT1 蛋白表达后,HCMV 感染组EA.hy926 细胞迁移(P = 0.008,0.003)和成管能力(P = 0.012,0.008)下降或增强;而细胞增殖能力无变化(P = 0.969,0.948).结论 HCMV 感染血管内皮细胞后能够促进细胞增殖、迁移和小管生成能力,其作用机制可能与抑制SIRT1蛋白表达有关.%Background Human cytomegalovirus (HCMV) is closely related to diseases including atherosclerosis, transplanted vascular sclerosis and arterial restenosis. It has been proved that angiogenesis induced by HCMV infection could result in vascular diseases. This article aims to investigate the mechanism of angiogenesis induced by HCMV infection in endothelial cells. Methods Endothelial cells (EA.hy926 cells) were divided into HCMV infected group and mock infected group. The cells were collected at 2, 6, 12 and 24 h after infection. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to analyze SIRT1 mRNA and protein levels. Endothelial cells were incubated respectively by Resveratrol and SIRT1 small interference RNA (siRNA) for 2 h before infection. Twenty-four hours after infection, the proliferation, migration and tubule formation of cells were assessed by CCK-8, migration assay and tubule formation assay to detect the angiogenic response of endothelial cells. Results Compared to mock infected group, the expression of SIRT1 mRNA in HCMV infected group remained unchanged (F = 1.395, P = 0.304), but the expression of SIRT1 protein decreased gradually (F = 23.927, P = 0.000). Under the treatment of Resveratrol and SIRT1 siRNA, migration (P = 0.008, 0.003) and tubule formation (P = 0.012, 0.008) of endothelial cells increased or reduced. The proliferation, however, remained unchanged (P = 0.969, 0.948). Conclusion HCMV infection promotes proliferation, migration and tubule formation of endothelial cells, and its mechanism may be related to the supression of SIRT1.