17 ALPHA HYDROXYLASE EXPRESSION IN HUMAN OVARIAN CELLS

17 人卵巢细胞中的α羟化酶表达

• 批准号: 2673942
• 负责人: Jan M McAllister
• 金额: $ 17.09万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2673942
• 关键词: androgens; cyclic AMP; cytochrome P450; female; gel mobility shift assay; gene expression; gonadotropins; graafian follicles; growth factor; human tissue; messenger RNA; nuclear runoff assay; polycystic ovary syndrome; posttranscriptional RNA processing; protein biosynthesis; protein sequence; steroid 17alpha monooxygenase; steroid hormone biosynthesis; steroid hormone metabolism; tissue /cell culture

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.

描述（改编自研究者的摘要）：本研究将 研究人细胞色素 P450 17-α-羟化酶的调节 正常卵巢卵泡膜内细胞 (CYP17) 基因表达 骑自行车的女性和患有多囊卵巢综合症的女性的卵巢 （多囊卵巢综合症）。 要检验的假设是雄激素产生增加 由 PCOS 卵泡膜类固醇生成的内在异常引起 细胞。 目标是了解 LH 和生长因子如何调节 CYP17 正常细胞中的表达和雄激素合成，以及如何失调 这些过程会导致多囊卵巢综合症患者雄激素的产生增加。 已开发出繁殖正常细胞功能性培养物的条件 和 PCOS 卵泡膜细胞表达 17a-羟化酶活性以响应 营。 在正常培养的卵泡膜细胞中，cAMP 刺激 CYP17 mRNA，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) 抑制 CYP17 mRNA 的 cAMP 刺激。 该研究将描述 CYP17 基因表达的机制 受 LH 刺激，并受卵泡膜细胞生长因子抑制。 具体的 目标 1 将表征 CYP17 酶活性、蛋白质、 和膜培养物中的 mRNA 含量，并确定 LH 依赖性是否 CYP17 mRNA 的诱导需要持续的蛋白质合成。 这 研究人员将检查 LH 和 LH 的时间和剂量依赖性影响 生长因子对 CYP17 mRNA 水平的影响，并确定 mRNA 是否发生变化 稳定性也有助于 cAMP 和生长因子对 稳态 CYP17 mRNA。 此外，调查人员将确定是否 这些调节过程在多囊卵巢综合症中发生了改变。 具体目标 2 将 识别参与组织特异性的顺式调控元件 类固醇生成转录因子 LH 对 CYP17 基因的调节 因子 1 (SF-1) 和生长因子。 应调节生长因子 PCO 卵泡膜细胞中 CYP17 的表达发生改变，研究将开始 确定参与 CYP 17 调节的顺式调节元件 所涉及的特定生长因子的表达。 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物进行 以及对正常人和 PCOS 患者的卵泡膜细胞进行长期培养 检查类固醇生成途径的异常是否导致 多囊卵巢综合症患者雄激素分泌增加是外在的或内在的。 计划进行实验以确定雄激素的产生是否增加 LH 和生长因子调节变化的结果 P450 scc (CYP11A)、3B -HSD、CYP17 或某些组合的表达 这些在多囊卵巢综合症卵泡膜细胞中。 从这些研究中获得的信息将 更好地了解类固醇合成的分子基础 多囊卵巢综合症患者雄激素过多。