PRODUCTION & FUNCTION OF PDGF LIKE MOLECULES

最近的证据表明，一种类似于血小板衍生生长因子的 分子(PDGFc)是由几种正常的细胞类型在以下条件下产生的 与体内细胞增殖有关的环境。这 该项目将调查性质、规则和可能的角色 PDGFc.具体而言，该项目将解决以下问题： (1)血小板衍生生长因子与血小板衍生生长因子有区别吗？将对PDGFc进行比较 用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和等电泳法测定PDGF 凝胶后聚焦检测受体结合活性 抗PDGF免疫印迹洗脱及抗原反应性的研究 抗血清。(2)哪些细胞在什么情况下产生PDGFc？ 利用培养细胞，初步观察发育调节 大鼠主动脉平滑肌细胞(SMC)产生PDGFc，以及 凝血酶刺激血管内皮细胞产生PDGFc， 将进一步研究并推广到其他物种和细胞类型。 将使用生化和组织化学技术来研究PDGFc 活体生产。(3)PDGFc的生产是否在 转录、翻译、翻译后加工还是分泌？ 这将在使用凝血酶刺激的培养中进行研究 幼鼠和幼鼠内皮细胞与发育调控的差异 成年大鼠SMC作为模型系统。信使核糖核酸水平将由 与sis cdna探针杂交；潜在的蛋白质前体将是 免疫沉淀法鉴定细胞内[35]半胱氨酸 蛋白质和多肽特异性抗血清免疫印迹；活性PDGFc 将通过放射受体(RRA)进行定量。(4)是指 产生PDGFc的细胞上PDGF受体的数量 内源性受体合成减少或下调 合成的PDGFc？如果是后者，PDGFc受体在哪里？ 互动发生了吗？将开发技术来研究增溶 受体和PDGF-受体复合体，并测定 络合物的积累、形成的轨迹和生理的 细胞表面形成的后果对细胞内复合体的影响。(5) PDGFc的产生在决定表型中起重要作用吗？ 是PDGF反应细胞的一部分，还是仅仅是一种偶然现象？它的重要性 暴露于PDGFc的自分泌的胞外相将被评估 通过用PDGF抗体或PDGF抑制剂孵育测试细胞 PDGF结合。明确终止PDGFc合成的影响将是 通过用构建的载体转染PDGFc产生细胞进行评估 在可诱导启动子的控制下表达反义mRNA。

Recent evidence suggests that a platelet-derived growth factor-like molecule (PDGFc) is produced by several normal cell types under circumstances which are associated with cell proliferation in vivo. This project will investigate the nature, regulation, and possible roles of PDGFc. Specifically, the project will address the following questions: (1) Does PDGFc differ from PDGF from platelets? PDGFc will be compared with PDGF by SDS-polyacrylaminde gel electrophoresis and isoelectric focusing followed by detection of receptor-binding activity after gel elution and of antigenic reactivity by immunoblotting with anti-PDGF antisera. (2) What cells produce PDGFc and under what circumstances? Using cultured cells, initial observations of developmentally regulated production of PDGFc production by rat aortic smooth muscle cells (SMC), and of thrombin-stimulated production of PDGFc by vascular endothelial cells, will be further investigated and extended to other species and cell types. Biochemical and histochemical techniques will be used to investigate PDGFc production in vivo. (3) Is production of PDGFc regulated at the level of transcription, translation, post-translational processing, or secretion? This will be investigated in culture using thrombin stimulation of endothelial cells and developmentally regulated differences between pup and adult rat SMC as model systems. mRNA levels will be determined by hybridization with sis cDNA probes; potential protein precursors will be identified by immunoprecipitation of [35]cysteine-labeled intracellular proteins and by immunoblotting with peptide-specific antisera; active PDGFc will be quantitated by radioreceptor (RRA). (4) Is the reduction in the number of PDGF receptors on cells which produce PDGFc a consequence of decreased receptor synthesis or of downregulation by endogenously synthesized PDGFc? If the latter, where does the PDGFc-receptor interaction occur? Techniques will be developed to study solubilized receptors and PDGF-receptor complexes, and to determine the rate of accumulation of complexes, the locus of formation, and the physiological consequences of formation of cell surface vs intracellular complexes. (5) Does PDGFc production play an important role in determining the phenotype of a PDGF-responsive cell, or is it just an epiphenomenon? The importance of the extracellular phase of autocrine exposure to PDGFc will be evaluated by incubating the test cells with antibodies to PDGF or with inhibitors of PDGF binding. The effects of specifically terminating PDGFc synthesis will be evaluated by transfecting PDGFc-producing cells with a construct expressing anti-sense mRNA under the control of an inducible promotor.