PRODUCTION & FUNCTION OF PDGF LIKE MOLECULES

最近的证据表明，血小板衍生生长因子样 PDGFc分子（PDGFc）由几种正常细胞类型在 这些情况与体内细胞增殖有关。 这 该项目将调查的性质，监管和可能的作用， PDGFc。 具体而言，该项目将解决以下问题： (1)PDGFc与血小板中的PDGF不同吗？ 将比较PDGFc 用聚丙烯酰胺凝胶电泳和等电 凝胶后聚焦，然后检测受体结合活性 通过用抗PDGF免疫印迹法洗脱和检测抗原反应性 抗血清 (2)什么细胞产生PDGFc，在什么情况下产生？ 使用培养的细胞，初步观察到发育调节的 大鼠主动脉平滑肌细胞（SMC）产生PDGFc，和 凝血酶刺激血管内皮细胞产生PDGFc， 将进一步研究并扩展到其他物种和细胞类型。 生物化学和组织化学技术将用于研究PDGFc 体内生产。 (3)PDGFc的生产是否受到以下水平的监管： 转录、翻译、翻译后加工还是分泌？ 这将在培养中使用凝血酶刺激来研究。 内皮细胞和发育调节的差异之间的小狗和 成年大鼠SMC作为模型系统。 mRNA水平将通过 与siscDNA探针杂交;潜在蛋白前体将 通过免疫沉淀[35]半胱氨酸标记的细胞内 蛋白质，并用肽特异性抗血清进行免疫印迹;活性PDGFc 将通过放射受体（RRA）进行定量。 (4)是在减少 产生PDGFc的细胞上的PDGF受体的数量， 受体合成减少或内源性下调 合成PDGFc？ 如果是后者，PDGFc受体在哪里？ 互动发生？ 将开发技术来研究溶解的 受体和PDGF-受体复合物，并确定 复合物的积累，形成的场所，和生理 细胞表面与细胞内复合物形成的后果。 （五） PDGFc的产生是否在决定表型中起重要作用 还是只是一种附带现象 的重要性 将评价PDGFc自分泌暴露的细胞外相的 通过将测试细胞与PDGF的抗体或PDGF的抑制剂一起孵育， PDGF结合。 特异性终止PDGFc合成的效果将 通过用构建体转染产生PDGFc的细胞来评价 在诱导型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.