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， 心血管疾病发病率和死亡率相当高。 最近的进展已经 在我们对生长因子基因表达的理解中， 在体外调控，但一个主要的障碍，设计合理的 治疗学是生长因子基因表达的表征， 在体内发挥作用。 在本提案中，我们使用一种新颖的方法来提供 生物活性基因产物到动脉中的血管细胞 通过在体内直接转移重组基因的循环。 这将 允许选择性引入生长因子，所述生长因子的表达 和功能的基因产物的设计， 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 复制缺陷型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， 血管壁。 这些技术现在将得到优化，并用于 引入重组生长因子基因。 具体来说，我们将介绍 重组血小板源性生长因子（PDGF）B基因导入猪 髂股动脉，并确认其表达在体内通过分析 DNA转移（聚合酶链反应）、mRNA表达（原位 杂交、S1核酸酶保护、RNA PCR）、蛋白质表达 （免疫组织化学）和形态测定分析。 二是 确定重组PDGF B基因表达是否会刺激 细胞增殖和动脉粥样硬化病变的形成，或 与其它因子如PDGF AA、酸性和碱性FGF 和TGF β或与高脂血症相关。 三、基因产品 将构建当在体内表达时可抑制细胞增殖的 通过拮抗有丝分裂原与受体的结合而增殖，或 破坏基因调控。 这类抑制剂包括合成 编码猪相容性单克隆抗体的表达载体， 来自杂交瘤系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，并且对PDGF的影响 表达将被确定。 在初步实验中，我们有 建立了体内细胞增殖模型， 用重组PDGF B基因转染猪动脉。 正在确认重组PDGF基因表达， 将构建PDGF的拮抗剂。 总之，这些研究将 研究重组PDGF B的体内表达和功能 基因和其它重组生长因子基因。 这些研究结果可用于设计合理的， 治疗动脉粥样硬化的新的治疗方法， 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.