NOVEL HEPARIN-BINDING GROWTH FACTOR IN UTERINE FLUID

这项工作的广泛的、长期的目标是阐明 和调节生长发育的子宫分泌生长因子的功能 胚泡的胚外膜和/或增殖 子宫内膜上皮或间质。这项建议的重点是 一种新的生长因子在猪子宫腔冲洗中的特性 在怀孕早期，这一水平会升高。这个因素，阳离子，10,000-mr， 不耐热、不耐酸的丝裂原用于成纤维细胞、平滑肌和 子宫内膜细胞，一直被称为“肝素结合生长因子”。 本项目的具体目标是：1)从分子水平上分离 表征HBGF-0.8；2)分析其性质和功能 肝素-HBGF-0.8相互作用的意义；3)HBGF-0.8的定量 4)研究HBGF-0.8受体的定位和 在子宫中起作用。这个项目与健康的相关性 HBGF-0.8可以(I)促进囊胚发育，降低 宫内或体外受精后胚胎死亡的发生率 和(Ii)刺激周期子宫内膜重塑或有助于 子宫癌的发生和发展。本研究的设计与实现 制备柱纯化HBGF-0.8至均一的方法 层析(如阳离子交换、肝素亲和、反相) 并通过以下方式直接或间接确定该氨基酸序列 克隆并测序ITS基因。纯化的HBGF-0.8将被标记为 I研究(I)其与细胞外类肝素分子的结合 基质和细胞表面(II)其特殊高亲和力的性质 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。其效果 肝素在调节HBGF-0.8上的作用将通过确定哪个 人工合成的HBGF-0.8多肽与肝素结合并调节HBGF-1的结合 肝素为0.8。将用合成的多肽免疫兔子 与一个混杂的破伤风类毒素T细胞表位结合。特定的 用酶联免疫吸附试验筛选抗HBGF-0.8抗体。抗血清将会是 用于筛选HBGF-0.8的重组Gammagt11，以进行免疫定位 子宫切片中HBGF-0.8的含量，以定量HBGF-0.8在子宫组织中的竞争 发情、怀孕或类固醇处理的猪的子宫冲洗，以及 免疫印迹法检测HBGF-0.8。

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.