17 ALPHA HYDROXYLASE EXPRESSION IN HUMAN OVARIAN CELLS

描述（改编自研究者摘要）：本研究将 研究人细胞色素P450 17-α-羟化酶的调节 CYP 17基因在正常人卵巢内膜细胞中的表达 多囊卵巢综合征的病因有哪些 （PCOS）。 有待检验的假设是， PCOS卵泡膜中类固醇产生的内在异常导致 细胞 目的是了解LH和生长因子如何调节CYP 17 表达和雄激素的合成，以及如何失调， 这些过程导致PCOS中雄激素产生增加。 已经开发了条件来繁殖正常细胞的功能性培养物， 和PCOS卵泡膜细胞，其表达17 α-羟化酶活性以响应 营 在正常培养的卵泡膜细胞中，cAMP刺激CYP 17 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cAMP对CYP 17 mRNA的刺激。 这项研究将描述CYP 17基因表达的机制， 卵泡膜细胞中LH刺激，生长因子抑制。 具体 目的1将表征CYP 17酶活性、蛋白质、 和mRNA含量，并确定LH依赖性 CYP 17 mRNA的诱导需要持续的蛋白质合成。 的 研究者将检查LH的时间和剂量依赖性效应， 生长因子对CYP 17 mRNA水平的影响，并确定mRNA 稳定性也有助于cAMP和生长因子对 稳态CYP 17 mRNA。 此外，研究人员将确定是否 这些调节过程在PCOS中发生改变。 具体目标2将 确定参与组织特异性表达的顺式调节元件， LH对CYP 17基因的调节，LH是类固醇生成的转录因子 因子-1（SF-1）和生长因子。 应生长因子的调节 如果PCOS卵泡膜细胞中CYP 17的表达发生改变，研究将开始， 鉴定参与调节B17的顺式调节元件 由所涉及的特定生长因子表达。 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 长期培养正常和PCOS患者卵泡膜细胞， 检查类固醇生成途径的异常是否会导致 PCOS中雄激素产生增加是外源性的还是内源性的。 计划进行实验，以确定增加雄激素的产生是否 由LH和生长因子调节的变化引起， 表达P450 SCC（CYP 11 A）、3B-HSD、CYP 17或它们的某种组合 PCOS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 和雄激素过多

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.