17 ALPHA HYDROXYLASE EXPRESSION IN HUMAN OVARIAN CELLS

描述(改编自调查人员摘要)：这项研究将 人细胞色素P450 17-α-羟基酶的调控研究 (CYP17)基因在正常卵巢内膜细胞中的表达 骑自行车的妇女和多囊卵巢综合征妇女的卵巢 (PCOS)。需要检验的假设是，雄激素的产生增加 多囊卵巢综合征卵泡膜类固醇分泌的内在异常 细胞。目的是了解促黄体生成素和生长因子是如何调节细胞色素P17的。 正常细胞中雄激素的表达和合成，以及如何失调 这些过程导致多囊卵巢综合征中雄激素的产生增加。 已经开发了条件来繁殖正常的功能培养物 和表达17a-羟基酶活性的PCOS卵泡膜细胞 夏令营。在培养的正常卵泡膜细胞中，cAMP刺激细胞色素P17 m RNA，而 表皮生长因子(EGF)、成纤维细胞生长因子(FGF)和 转化生长因子B(TGFb)可抑制cAMP刺激的细胞色素P17基因表达。 这项研究将表征CYP17基因表达的机制。 卵泡膜细胞受促黄体生成素刺激，生长因子抑制。特定的 目的1将表征细胞色素P17酶活性、蛋白质、 和卵泡膜培养物中的mRNA含量，并确定是否依赖于促黄体生成素 诱导细胞色素P17基因的表达需要持续的蛋白质合成。这个 研究人员将研究黄体生成素和黄体生成素的时间和剂量依赖效应。 生长因子对细胞色素P17基因表达水平的影响，并决定其基因表达是否发生变化 稳定性也有助于cAMP和生长因子对 稳态细胞色素P17基因的表达。此外，调查员将确定是否 这些调控过程在多囊卵巢综合征中发生了改变。特定目标2将 确定组织特异性中涉及的顺式调控元件 类固醇合成转录因子黄体生成素对细胞色素P17基因的调控 因子-1(SF-1)和生长因子。应该对生长因子进行调节 CyP17在PCOS膜细胞中的表达被改变，研究将开始 确定参与CYP 17调控的顺式调控元件 通过所涉及的特定生长因子来表达。在具体目标3中， 类固醇代谢的研究将用新鲜的外植体培养进行。 从正常和PCOS患者的卵泡膜细胞长期培养到 检查类固醇合成途径的异常是否会导致 多囊卵巢综合征患者雄激素分泌增加是外源性的或内在的。 计划进行实验，以确定增加雄激素的产生 促黄体生成素和生长因子调节的变化 P450 SCC(CyP11A)、3B-HSD、CyP17或其组合的表达 这些存在于PCOS卵泡膜细胞中。从这些研究中获得的信息将 更好地理解类固醇合成的分子基础 多囊卵巢综合征中雄激素过多。

DESCRIPTION (Adapted from the Investigator's Abstract): This study will investigate the regulation of human cytochrome P450 17-alpha- hydroxylase (CYP17) gene expression in theca interna cells from ovaries of normal cycling women and from ovaries of women with polycystic ovarian syndrome (PCOS). The hypothesis to be tested is that increased androgen production results from an intrinsic abnormality of steroid production in PCOS theca cells. The goal is to understand how LH and growth factors regulate CYP17 expression and androgen synthesis in normal cells, and how dysregulation of these processes results in increased androgen production in PCOS. Conditions have been developed to propagate functional cultures of normal and PCOS theca cells that express 17a-hydroxylase activity in response to cAMP. In normal, cultured theca cells, cAMP stimulates CYP17 mRNA, whereas epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor B (TGFB) inhibit cAMP-stimulation of CYP17 mRNA. The study will characterize the mechanisms by which CYP17 gene expression is stimulated by LH, and inhibited by growth factors in theca cells. Specific Aim 1 will characterize the regulation of CYP17 enzyme activity, protein, and mRNA content in theca cultures, and determine whether the LH-dependent induction of CYP17 mRNA requires ongoing protein synthesis. The investigator will examine the time-and dose-dependent effects of LH and growth factors on CYP17 mRNA levels, and determine whether changes in mRNA stability also contribute to the effects of cAMP and growth factors on steady state CYP17 mRNA. Moreover, the investigator will determine whether these regulatory processes are altered in PCOS. Specific Aim 2 will identify the cis- regulatory elements involved in the tissue-specific regulation of CYP17 gene by LH, the transcription factor steroidogenic factor-1 (SF-1), and growth factors. Should growth factor modulation of CYP17 expression in PCOs theca cells be altered, studies will begin to identify the cis-regulatory elements involved in the regulation of CYP 17 expression by the specific growth factor involved. In Specific Aim 3, studies of steroid metabolism will be performed with fresh explant cultures and long-term cultures of theca cells from normal and PCOS patients to examine whether the abnormalities in the steroidogenic pathway that cause increased androgen production in PCOS are extrinsic or intrinsic. Experiments are planned to determine whether increased androgen production results from changes in the regulation by LH and growth factors of expression of P450 scc (CYP11A), 3B -HSD, CYP17, or some combination of these in PCOS theca cells. Information derived from these studies will provide a better understanding of the molecular basis of steroid synthesis and androgen excess in PCOS.