目的 长单一序列区(unique long region 83,UL83)基因是人巨细胞病毒(human cytomegalovirus,HCMV)感染再激活标志.本研究拟探索UL83对HCMV感染后人脑血管平滑肌细胞(vascular smooth muscle cel s,VSMC)中不稳定斑块破裂相关因子,如促血管生成素(angiopoietin,Ang)以及血管内皮生长因子(vascular endothelial growth factor,VEGF)的影响.方法 ①采用流式细胞仪确定amidite荧光素(fluorescein amidite,FAM)标记的小干扰核糖核酸(smal interfering ribonucleic acid,siRNA)通过脂质体转入人脑VSMC的转染效率;②将3条化学合成siRNA通过脂质体转染入已感染HCMV AD169株(MOI=10)的人脑VSMC当中,结合空白组、接毒组及接毒无效干扰组筛选抑制UL83基因最强siRNA;③分别检测HCMV AD169株(MOI=10)感染人脑VSMC后0 h、6 h、12 h、24 h、48 h以及72 h Ang-1、Ang-2及VEGF-A信使核糖核酸(messenger ribonucleic acid,mRNA)及蛋白表达变化;④分别检测空白组、接毒高效干扰组、接毒无效干扰组及接毒组在感染48 h后Ang-1、Ang-2及VEGF-A的mRNA及感染72 h后上述蛋白表达情况.结果 转染6 h后,95.59％人脑VSMC转染了FAM标记siRNA.感染HCMV AD169株(MOI=10)的人脑VSMC在转染siRNA 48 h后,siRNA转染组与接毒组相比,UL83 mRNA相对表达量最多下降78.7％;转染siRNA72 h后,UL83编码的pp65蛋白相对表达量最多下降81.3％.人脑VSMC感染HCMV AD169株(MOI=10)0 h、6 h、12 h、24 h、48 h以及72 h时后,Ang-1 mRNA及蛋白相对表达量逐渐下降(P<0.01),Ang-2及VEGF-A mRNA及蛋白相对表达量则逐渐增加(P<0.01).而通过转染高效siRNA抑制HCMV AD169株UL83基因,使得Ang-1表达下降,Ang-2和VEGF-A表达增加均被抑制.结论 HCMV AD169株具有同时促进人脑VSMC中Ang-1表达降低,Ang-2及VEGF-A表达增加功能.而通过siRNA抑制HCMV AD169株UL83基因表达能够阻止上述变化.提示HCMV可能通过UL83基因造成靶细胞产生促血管新生微环境.%Objective Unique long region 83 (UL83) gene is the symbol of human cytomegalovirus (HCMV) reactivated infection. This study is to investigate relationships between UL83 and vulnerable plaques rupture related factors, including angiopoietin (Ang) and vascular endothelial growth factor (VEGF) in HCMV infected human brain vascular smooth muscle cells (HBVSMCs). Methods (1) Cytoflowmetry was used to identify the efficiency of fluorescein amidite (FAM) labeled small interfering RNA (siRNA) transfection into HBVSMCs. (2) Three groups of siRNA were transfected into HBVSMCs infected with HCMV AD169 (MOI=10) in advance, and combinedwith groups of blank, infected and infected but ineffective interfered to find which siRNA could silence UL83 gene to the greatest extent. (3) The messenger ribonucleic acid (mRNA) and protein expressions of Ang-1, Ang-2 and VEGF-A were detected at different time from 0, 6, 12, 24, 48 until 72 hours after HBVSMCs infected with HCMV AD169 (MOI=10), respectively. (4) mRNA and protein expressions of Ang-1, Ang-2 and VEGF-A in blank, infected & effective interfered, infected but ineffective interfered and only infected HBVSMCs were compared. Results A total of 95.95％ of HBVSMCs had been transfected with FAM labelled siRNA after 6 hours. Forty-eight hours after transfected with siRNA, relative expression of UL83 mRNA within HBVSMCs infected with HCMV AD169 (MOI=10) reduced 78.7％ at most, while 72 hours after transfected with siRNA, relative expression of UL83 encoded pp65 protein reduced 81.3％ at most. Relative mRNA and protein expressions of Ang-10, 6, 12, 24, 48 and 72 hours after HBVSMCs infected with HCMV AD169 (MOI=10) gradually reduced (P<0.01), while relative mRNA and protein levels of Ang-2 and VEGF-A gradually increased (P<0.01). After silencing UL83 gene with effective siRNA, both reduction of Ang-1 expression and increase of Ang-2, VEGF-A expressions had been halted. Conclusion HCMV AD169 can reduce Ang-1 expression but increase Ang-2, VEGF-A expressions at the same time. Silence HCMV AD169 UL83 gene expression may halt such trend, which suggests the potential capability of HCMV making target cells producing angiogenic microenvironment through UL83 gene.
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