EXPRESSION AND BIOLOGICAL FUNCTION OF RECOMBINANT PDGF

血管损伤后的细胞增殖有助于 动脉粥样硬化和再狭窄这两种临床疾病 相当大的心血管发病率和死亡率。最近的进展已经 在我们对生长因子基因表达和 体外调控，然而理性设计的一大障碍 治疗学是对生长因子基因表达和 在体内发挥作用。在这份提案中，我们使用了一种新的方法来交付 动脉血管细胞的生物活性基因产物 通过在体内直接转移重组基因进行循环。这将是 允许选择性地引入生长因子(S)，其表达 和功能可以表征的基因产品的设计 可能调节和抑制这种生长因子基因的表达。我们有 最近在这一领域取得了重大进展，表明 复制缺陷逆转录病毒载体或脂质体可用于 通过直接转导将报告基因导入动脉部位 脉管壁。这些技术现在将被优化并用于 引入重组生长因子基因。具体地说，我们将推出 重组血小板衍生生长因子B基因导入猪的研究 并通过分析确定其在体内的表达。 DNA转移(聚合酶链式反应)、mRNA表达(原位 杂交、S1核酸酶保护、RNA聚合酶链式反应、蛋白质表达 (免疫组织化学)和形态计量学分析。第二，我们将 确定重组PDGF B基因表达是否会刺激 单纯动脉粥样硬化性病变的细胞增殖和形成 结合其他因素，如PDGF AA、酸性和碱性成纤维细胞生长因子， 和转化生长因子β或与高脂血症相关。第三，基因产品 在体内表达时可能会抑制细胞 通过拮抗有丝分裂原与受体或受体的结合而增殖 扰乱基因调控。这类抑制剂包括合成 编码猪相容单抗的表达载体 杂交瘤细胞株的生长因子和可溶性受体。这些 潜在的抑制剂将在体内表达，并对PDGF的影响 表达方式将会确定。在初步实验中，我们有 建立了一种体内细胞增殖模型，该模型由 重组PDGF B基因导入猪动脉的实验研究 正在进行重组PDGF基因表达的确认，以及 将构建PDGF的拮抗剂。总而言之，这些研究将 重组人血小板衍生生长因子B的体内表达及功能研究 基因和其他重组生长因子基因的直接基因转移。 这些研究的结果可能对设计Rational和 治疗动脉粥样硬化和高血压的新方法 血管再狭窄。

Cellular proliferation following vascular injury contributes to atherosclerosis and restenosis, two clinical diseases which account for considerable cardiovascular morbidity and mortality. Recent advances have been made in our understanding of growth factor gene expression and regulation in vitro, yet a major obstacle to the design of rational therapeutics is the characterization of growth factor gene expression and function in vivo. In this proposal, we use a novel approach to deliver biologically active gene products to vascular cells in the arterial circulation by direct transfer of recombinant genes in vivo. This will permit the selective introduction of a growth factor(s) whose expression and function can be characterized and the design of gene products which might regulate and inhibit such growth factor gene expression. We have recently made significant progress in this area, having shown that replication-defective retroviral vectors or liposomes can be used to introduce a reporter gene into an arterial site by direct transduction of the vessel wall. These techniques will now be optimized and used to introduce recombinant growth factor genes. Specifically, we will introduce the recombinant platelet derived growth factor (PDGF) B gene into porcine iliofemoral arteries and confirm its expression in vivo by analysis of transfer of DNA (polymerase chain reaction), expression of mRNA (in situ hybridization, S1 nuclease protection, RNA PCR), expression of protein (immunohistochemistry), and morphometric analysis. Second, we will determine whether recombinant PDGF B gene expression will stimulate cellular proliferation and formation of an atherosclerotic lesion alone or in combination with other factors, such as PDGF AA, acidic and basic FGF, and TGF beta or in association with hyperlipidemia. Third, gene products will be constructed which when expressed in vivo might inhibit cellular proliferation by antagonizing the binding of mitogen to receptor or disrupting gene regulation. Such inhibitors include the synthesis of expression vectors encoding porcine compatible monoclonal antibodies to growth factors from hybridoma lines, and soluble receptors. These potential inhibitors will be expressed in vivo, and effects on PDGF expression will be determined. In preliminary experiments, we have established a model of cellular proliferation in vivo, induced by transfection of porcine arteries with the recombinant PDGF B gene. Confirmation of recombinant PDGF gene expression is being performed, and antagonists to PDGF will be constructed. In summary, these studies will investigate the in vivo expression and function of the recombinant PDGF B gene and other recombinant growth factor genes using direct gene transfer. The results of these studies may be useful in the design of rational and novel therapeutic approaches to the treatment of atherosclerosis and restenosis.