ESTROGEN-INDUCED GROWTH FACTOR EXPRESSION IN THE UTERUS

子宫内雌激素诱导的生长因子表达

• 批准号: 3426700
• 负责人: ROBERT D KOOS
• 金额: $ 5万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3426700
• 关键词: RNA directed DNA polymerase; SDS polyacrylamide gel electrophoresis; angiogenesis factor; estrogens; fibroblast growth factor; gene expression; growth factor; hormone regulation /control mechanism; keratinocyte; laboratory rat; messenger RNA; mitogens; polymerase chain reaction; subtraction hybridization; uterus; vascular endothelium

The long term goal of this study is to determine how estrogen stimulates growth of the mammalian uterus. Estrogen stimulates hypertrophy and hyperplasia of the uterus and other organs of the female genital tract. This effect is mediated directly at the target tissue via interaction of estrogen with its specific receptor. It is believed that many of estrogen's effects are mediated by growth regulatory factors whose synthesis and/or activity are regulated by estrogen. Despite the significance of these processes to human fertility, relatively little is known about the nature of these factors. In this study, the powerful, sensitive method of reverse transcription-polymerase chain reaction (RT-PCR) will be used to evaluate estrogen's effects on the expression of growth regulatory factors in the rat uterus. In part 1 (Specific Aim I), the ability of estrogen to control the expression of four growth factors that seem likely to play important roles in the cyclic development of the uterus, but whose expression in the uterus is almost completely unexplored, will be determined. These include: 1) acidic fibroblast growth factor (FGF), 2) basic FGF, 3) keratinocyte growth factor (KGF), and 4) vascular endothelial growth factor (VEGF). The FGF's are broad spectrum mitogens that stimulate proliferation of a wide range of cells and are thought to be involved in neovascularization. Expression of basic FGF by human endometrial adenocarcinoma cells has been shown to be stimulated by estrogen. KGF is similar in structure to the FGF's but is specific for epithelial cells; KGF is thought to be a stromally-derived regulator of epithelial cell proliferation. It could, therefore, play a role in the proliferation of endometrial epithelium. VEGF is similar in structure to platelet derived growth factor (PDGF). It is a potent, specific endothelial cell mitogen; in addition it increases vascular permeability. VEGF could play a role in the development and functioning of the uterine vasculature. RNA will be extracted from uteri of control and estrogen-treated immature rats at various intervals after treatment and the expression of the mRNA's for these factors quantitatively analyzed. In part 2 (Specific Aim 2), the induction of as yet undefined mRNA's by estrogen in the uterus will be examined. This will be done using the method of subtraction hybridization followed by PCR amplification of the subtracted mRNA's. To do this, a defined DNA adaptor is ligated to both ends of the products a cDNA synthesis reaction performed on the subtracted mRNA's followed by standard PCR amplification, sequencing of the amplified products, and comparison of the sequences to known genes. In this way, low abundance mRNA's induced by estrogen may be identified; most growth factor mRNA's are relatively rare. These studies will provide new insight into the nature of the growth regulating factors involved in sex steroid hormone action on the female genital tract.

这项研究的长期目标是确定雌激素是如何刺激 哺乳动物子宫的生长。雌激素刺激肥大， 子宫和女性生殖道其他器官的增生。 这种作用通过以下相互作用直接在靶组织介导： 雌激素及其特异性受体。 据信，许多 雌激素的作用是由生长调节因子介导的， 合成和/或活性受雌激素调节。 尽管 这些过程对人类生育力的重要性，相对来说很少 了解这些因素的性质。 在这项研究中， 逆转录-聚合酶链反应 采用RT-PCR方法检测雌激素对人乳腺癌细胞中 大鼠子宫中的生长调节因子。 在第1部分（具体目标一）中， 雌激素控制四种生长因子表达的能力 似乎可能在循环发展中发挥重要作用， 子宫，但其在子宫中的表达几乎完全未被探索， 将被确定。这些包括：1）酸性成纤维细胞生长因子 (FGF)2）碱性FGF，3）角质形成细胞生长因子（KGF），和4）血管内皮生长因子（VEGF）。 内皮生长因子（VEGF）。 FGF是广谱的有丝分裂原 刺激多种细胞的增殖， 参与新生血管形成。 人碱性成纤维细胞生长因子的表达 子宫内膜腺癌细胞已经被证明是刺激的 雌激素. KGF在结构上与FGF类似，但特异于 上皮细胞; KGF被认为是一种基质衍生的调节因子， 上皮细胞增殖 因此，它可以在以下方面发挥作用： 子宫内膜上皮增生。 VEGF在结构上类似于 血小板衍生生长因子（PDGF）。它是一种有效的特异性内皮细胞 细胞有丝分裂原;此外，它增加血管通透性。 VEGF可以 在子宫脉管系统的发育和功能中发挥作用。 将从对照组和雌激素处理的未成熟小鼠的子宫中提取RNA 大鼠在治疗后的不同时间和mRNA的表达 对这些因素进行了定量分析。 在第2部分（具体目标2）中， 子宫内雌激素对尚未确定的mRNA的诱导将是 考察 这将使用减法杂交的方法来完成 随后PCR扩增减去的mRNA。 为此，A 将确定的DNA接头连接到产物cDNA的两端 在减去的mRNA上进行合成反应，然后进行标准的 PCR扩增，扩增产物测序，和比较 已知基因的序列 以这种方式，低丰度mRNA的诱导 雌激素可以被鉴定;大多数生长因子mRNA相对罕见。 这些研究将提供新的洞察增长的性质 性类固醇激素对雌性作用的调节因子 生殖道