Comparison of gene silencing in human vascular cells using small interfering RNAs
Comparison of gene silencing in human vascular cells using small interfering RNAs
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DOI：
10.1016/j.jamcollsurg.2006.12.029
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发表时间：
2007-03-01
影响因子：
5.2
通讯作者：
LoGerfo, Frank W.
中科院分区：
文献类型：
--
作者：
Andersen, Nicholas D.;Monahan, Thomas S.;LoGerfo, Frank W.
背景：通过小干扰RNA(SiRNA)转基因实现的基因沉默是血管基因治疗的一种很有前途的方法。在此，我们通过比较单基因和多基因siRNA在体外对人血管细胞的RNA干扰(RNAi)反应来表征RNAi在血管生物学中的行为。研究设计：利用定量逆转录-聚合酶链式反应(QRT-PCR)和Western印迹技术，分别或同时将针对甘油醛-3-磷酸脱氢酶、肉豆蔻酰化富含丙氨酸激酶C底物的siRNA单独或同时转染培养的人冠状动脉平滑肌和人冠状动脉内皮细胞(HCASMC/HCAEC)，以评价多基因沉默的强度和特异性。结果：小剂量(0.25~10 nM)siRNA转染后，两种细胞均有明显的靶点沉默，但作用弱于单基因siRNA。HCAECs中多基因信使RNA(mRNA量)的减少超过HCASMCs，低剂量siRNA转染后HCAECs的mRNA量和siRNA转运量均高于HCAECs。4结论：siRNA抑制多基因沉默是一种很有前途的非病毒调控血管细胞基因表达的方法。在我们的体外条件下，内皮细胞比血管平滑肌细胞更容易受到siRNA转染和基因沉默的影响。核糖核酸！这项技术可能会被用于开发siRNA鸡尾酒，用于静脉搭桥移植，或者用于调节其他形式的血管疾病的内皮细胞功能。
BACKGROUND: Gene silencing achieved through small interfering RNA (siRNA) transfection represents a promising approach to vascular gene therapy. Here we characterize the behavior of RNA interference (RNAi) in vascular biology by comparing the RNAi response to single- and multigene siRNA transfections in vitro in human vascular cells.STUDY DESIGN: The strength and specificity of multigene silencing in cultured human coronary artery smooth muscle and human coronary artery endothelial cells (HCASMC/HCAEC) were assessed by quantitative reverse transcription-polymerase chain reaction (QRT-PCR) and Western blot after transfection singly or simultaneously with siRNAs targeting glyceraldehyde-3-phosphate dehydrogenase, the myristoylated alanine-rich C kinase substrate, and cadherin 11. RNAi response to low-dose (0.25 to 10 nM) siRNA transfection was characterized between the two cell types by QRT-PCR and fluorescence-activated cell sorter analysis.RESULTS: Powerful and specific silencing of all targets was observed in both cell types after multigene siRNA transfections, but with a reduction in effect compared with single-gene siRNA transfections. Multigene messenger RNA (mRNA) reductions in HCAECs exceeded those achieved in HCASMCs, and superior mRNA silencing and siRNA delivery were observed in HCAECs after low-dose siRNA transfections. \CONCLUSIONS: Multigene silencing by siRNA stands as a promising nonviral approach for manipulating gene expression in human vascular cells. Under our in vitro conditions, endothelial cells were more susceptible to siRNA transfection and gene silencing than vascular smooth muscle cells. RNA! technology could potentially find use in the development of siRNA cocktails for application to vein bypass grafts or for modulating endothelial cell function in other forms of vascular disease.