Shaw Lynn C, Afzal Aqeela, Lewin Alfred S, Timmers Adrian M, Spoerri Polyxenie E, Grant Maria B
Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, Florida 32610, USA.
Invest Ophthalmol Vis Sci. 2003 Sep;44(9):4105-13. doi: 10.1167/iovs.03-0295.
Insulin-like growth factor (IGF)-1 and its receptor (IGF-1R) are associated with abnormal retinal neovascularization. Ribozymes were designed that selectively decreased the expression of the IGF-1R and these ribozymes were tested in angiogenesis models in vitro and in vivo.
Two hammerhead ribozymes were designed that cleave the human IGF-1R mRNA. The ribozymes were cloned into recombinant adeno-associated viral vectors (rAAV). The rAAV constructs were transfected into human retinal endothelial cells (HRECs). IGF-1R mRNA and protein levels were examined and the modified Boyden chamber assay used to examine ribozyme effects on cell migration. These constructs were injected intravitreally into mice to determine the effect of the ribozymes on retinal neovascularization in a mouse model of oxygen-induced retinopathy.
Relative quantitative RT-PCR analysis showed that IGF-1R Rz1 reduced IGF-1R mRNA levels by 40% +/- 10% (P = 0.003), and Western blot analysis showed a 41% +/- 5% (P = 4.6 x 10(-5)) reduction of IGF-1R protein, confirming that this ribozyme reduces IGF-1R expression. IGF-1R Rz1 also reduced IGF-1-induced cell migration by 90% +/- 5% (P = 2.9 x 10(-9)) showing that IGF-1R Rz1 reduces IGF-1R function in HRECs. IGF-1R Rz1 also reduced the amount of preretinal neovascularization by 65% +/- 6% (P = 2.7 x 10(-5)), as measured by the average number of endothelial preretinal nuclei per section.
These studies demonstrate that the IGF-1R ribozymes are effective at reducing the expression and function of the IGF-1R in vitro and in vivo. Therefore, the IGF-1R ribozymes are an effective method for studying the process of angiogenesis and may ultimately be effective as gene therapy tools for the reduction of pathologic retinal angiogenesis.
胰岛素样生长因子（IGF）-1及其受体（IGF-1R）与异常视网膜新生血管形成相关。设计了能选择性降低IGF-1R表达的核酶，并在体外和体内血管生成模型中对这些核酶进行了测试。
设计了两种切割人IGF-1R mRNA的锤头状核酶。将核酶克隆到重组腺相关病毒载体（rAAV）中。将rAAV构建体转染到人视网膜内皮细胞（HREC）中。检测IGF-1R mRNA和蛋白水平，并使用改良的博伊登小室试验检测核酶对细胞迁移的影响。将这些构建体玻璃体内注射到小鼠体内，以确定核酶对氧诱导视网膜病变小鼠模型中视网膜新生血管形成的影响。
相对定量逆转录-聚合酶链反应分析显示，IGF-1R Rz1使IGF-1R mRNA水平降低了40%±10%（P = 0.003），蛋白质印迹分析显示IGF-1R蛋白降低了41%±5%（P = 4.6×10⁻⁵），证实该核酶可降低IGF-1R表达。IGF-1R Rz1还使IGF-1诱导的细胞迁移降低了90%±5%（P = 2.9×10⁻⁹），表明IGF-1R Rz1可降低HREC中IGF-1R的功能。通过每切片视网膜前内皮细胞核的平均数量测量，IGF-1R Rz1还使视网膜前新生血管形成量降低了65%±6%（P = 2.7×10⁻⁵）。
这些研究表明，IGF-1R核酶在体外和体内均能有效降低IGF-1R的表达和功能。因此，IGF-1R核酶是研究血管生成过程的有效方法，最终可能作为减少病理性视网膜血管生成的基因治疗工具发挥作用。
