NOVEL HEPARIN-BINDING GROWTH FACTOR IN UTERINE FLUID

子宫液中新型肝素结合生长因子

• 批准号: 2202660
• 负责人: DAVID R BRIGSTOCK
• 金额: $ 11.22万
• 依托单位: CHILDREN'S HOSPITAL (COLUMBUS)
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2202660
• 关键词: 3T3 cells; affinity chromatography; binding proteins; cell membrane; complementary DNA; embryo /fetus; endometrium; enzyme linked immunosorbent assay; extracellular matrix; fibroblast growth factor; genetic mapping; growth factor receptors; heparin; intermolecular interaction; laboratory rabbit; protein purification; protein sequence; protein structure function; receptor binding; secretion; swine; synthetic peptide; uterus; western blottings

The broad, long-term objectives of this work are to elucidate the nature and function of uterine secretory growth factors that regulate the growth of the extra-embryonic membranes of the blastocyst and/or proliferation of endometrial epithelium or stroma. This proposal focusses on the characterization of a novel growth factor in pig uterine luminal flushing that is elevated in early pregnancy. This factor, a cationic, 10,000-Mr, heat-labile, acid-labile mitogen for fibroblasts, smooth muscle and endometrial cells, has been termed "heparin-binding growth factors. The specific aims of this project are 1) TO isolate and molecularly characterize HBGF-0.8; 2) To analyze the nature and functional significance of heparin-HBGF-0.8 interactions; 3) To quantify HBGF-0.8 in uterine fluids; and 4) To study HBGF-0.8 receptor localization and function in the uterine tract. The health relatedness of this project is that HBGF-0.8 may (i) promote blastocyst development and reduce the incidence of embryonic mortality in utero or after in vitro fertilization and (ii) stimulate cyclic endometrial remodelling or contribute to the onset and progression of uterine tract cancers. The research design and methods are to purify HBGF-0.8 to homogeneity using preparative column chromatography (e.g. cation exchange, heparin-affinity, reverse-phase) and to determine this amino acid sequence directly or indirectly by cloning and sequencing its cDNA. Purified HBGF-0.8 will be labelled with I to study (i) its binding to heparin-like molecules in extracellular matrix and cell surfaces (ii) the nature of its specific high affinity receptors and (iii) the presence of functional HBGF-0.8 receptors on freshly isolated or cultured endometrial or trophoderm cells. The effect of heparin on mediating HBGF-0.8 will be mapped by determining which synthetic HBGF-0.8 peptides bind to heparin and modulate binding of HBGF- 0.8 to heparin. Rabbits will be immunized with synthetic peptides that are conjugated to a promiscuous tetanus toxoid T-cell epitope. Specific anti-HBGF-0.8 antibodies will be selected by ELISA. Antisera will be used to screen recombinant gammagt11 for HBGF-0.8, to immunolocalize HBGF-0.8 in uterine sections, to quantify HBGF-0.8 competitive ELISA in uterine flushing from estrous, pregnant, or steroid-treated pigs, and for detecting HBGF-0.8 on Western blots.

这项工作的广泛的长期目标是阐明 和子宫分泌生长因子调节生长的功能 胚泡的胚外膜和/或增殖 子宫内膜上皮或间质。 该提案的重点是 一种新的生长因子在猪子宫腔冲洗中的特性 在怀孕早期会升高 这个因子，阳离子，10,000-Mr， 热不稳定、酸不稳定的成纤维细胞、平滑肌和 子宫内膜细胞，被称为“肝素结合生长因子”。 的 该项目的具体目标是1）分离和分子 对HBGF-0.8进行表征; 2）分析HBGF-0.8的性质和功能 肝素-HBGF-0.8相互作用的意义; 3）定量HBGF-0.8 研究HBGF-0.8受体在子宫液中的定位， 在子宫道中起作用。 该项目的健康相关性 HBGF-0.8可以（i）促进胚泡发育并减少 子宫内或体外受精后胚胎死亡率 和（ii）刺激周期性子宫内膜重塑或有助于 子宫道癌的发病和进展。 研究设计和 方法是用制备柱纯化HBGF-0.8至均一 色谱法（例如阳离子交换、肝素亲和、反相） 并通过直接或间接测定该氨基酸序列， 克隆并测序。 纯化的HBGF-0.8将标记为 I研究（i）其与细胞外肝素样分子的结合 基质和细胞表面（ii）其特异性高亲和力的性质 受体和（iii）功能性HBGF-0.8受体的存在 新鲜分离或培养的子宫内膜或滋养层细胞。 效果 肝素对介导HBGF-0.8的作用将通过确定 合成的HBGF-0.8肽与肝素结合并调节HBGF-0.8肽与肝素的结合。 0.8肝素。 将用合成肽免疫兔子， 与混杂的破伤风类毒素T细胞表位结合。 具体 通过ELISA选择抗HBGF-0.8抗体。 抗血清将 用于筛选HBGF-0.8的重组gammagt 11， 子宫切片中的HBGF-0.8，以定量子宫切片中的HBGF-0.8竞争性ELISA。 发情、妊娠或类固醇处理猪的子宫冲洗，以及 在Western印迹上检测HBGF-0.8。