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首页> 外文期刊>Endothelium: Journal of endothelial cell research >Approaches to enhance expression after adenovirus-mediated gene transfer to the carotid artery.
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Approaches to enhance expression after adenovirus-mediated gene transfer to the carotid artery.

机译：腺病毒介导的基因转移到颈动脉后增强表达的方法。

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The goal of this study was to enhance transgene expression after adenoviral-mediated gene transfer to the carotid artery. We used an adenoviral vector with a transgene that expresses beta-galactosidase, driven by the human cytomegalovirus (CMV) promoter/enhancer. The CMV promoter drives constitutive expression, and response elements within the enhancer allow inducible expression through binding of active transcription factors, such as cAMP response element binding protein (CREB) and nuclear factor kappa B (NFkappaB). Rings of rabbit carotid artery were incubated ex vivo with a replication-deficient adenovirus that expresses beta-galactosidase (AdCMV-betagal). Virus was removed from the medium, and forskolin or phorbol-12-myristate-13-acetate (PMA), which can induce activation of CREB or NFkappaB, respectively, were added to the medium. Pyrrolidine dithiocarbamate (PDTC) was used to inhibit activation of NFkappaB. Following incubation for 24 hours, beta-galactosidase activity was assessed by chemiluminescent reporter assay. Forskolin and PMA enhanced transgene expression in the carotid artery. Activity increased from 56+/-13 mU/mg protein (mean+/-SE) in rings of carotid treated with virus alone (10(9) pfu) to 159+/-23 mU/mg protein (P<0.05) in rings treated with forskolin, and to 189+/-40 mU/mg protein (P<0.05) in rings treated with PMA. Phorbol didecanoate, an inactive phorbol, did not affect expression of beta-galactosidase. After pre-incubation with PDTC prior to PMA, expression of beta-galactosidase was less than in rings incubated with PMA alone (29+/-11, P<0.05). Histochemical staining of carotid artery for beta-galactosidase demonstrated enhanced endothelial expression following administration of PMA. These findings suggest that expression after gene transfer to the carotid artery using an adenoviral vector with the CMV promoter/enhancer may be enhanced by PMA and forskolin, perhaps by activation of transcription factors.
机译：这项研究的目的是在腺病毒介导的基因转移到颈动脉后增强转基因表达。我们使用了带有转基因的腺病毒载体，该转基因表达由人巨细胞病毒（CMV）启动子/增强子驱动的β-半乳糖苷酶。 CMV启动子驱动组成型表达，而增强子中的响应元件则通过结合活性转录因子（例如cAMP响应元件结合蛋白（CREB）和核因子κB（NFkappB））来诱导表达。兔颈动脉环与表达β-半乳糖苷酶（AdCMV-βgal）的复制缺陷型腺病毒离体孵育。从培养基中除去病毒，并将分别可以诱导CREB或NFκB活化的福司柯林或佛波醇-12-肉豆蔻酸酯-13-乙酸酯（PMA）加入培养基中。使用吡咯烷二硫代氨基甲酸酯（PDTC）抑制NFkappaB的活化。温育24小时后，通过化学发光报告物测定法评估β-半乳糖苷酶活性。 Forskolin和PMA增强了颈动脉中的转基因表达。活性从仅用病毒（10（9）pfu）处理的颈动脉环中的56 +/- 13 mU / mg蛋白（平均+/- SE）增加到环中的159 +/- 23 mU / mg蛋白（P <0.05）用Forskolin处理后，在用PMA处理的环中达到189 +/- 40 mU / mg蛋白（P <0.05）。佛波二癸酸酯（一种无活性的佛波）不影响β-半乳糖苷酶的表达。在PMA之前与PDTC预温育后，β-半乳糖苷酶的表达低于仅与PMA温育的环中的表达（29 +/- 11，P <0.05）。给予PMA后，颈动脉的β-半乳糖苷酶组织化学染色显示内皮表达增强。这些发现表明，使用具有CMV启动子/增强子的腺病毒载体将基因转移至颈动脉后的表达可能通过PMA和福司可林增强，也许是通过激活转录因子来增强。

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