抗苗勒氏管激素调控干细胞因子抑制卵泡募集的分子机制

TO investigate the molecular mechanisms and extracellular signaling factors of follicle recruitment may ultimately prolong a female's reproductive lifespan. AS we known, anti-Mullerian hormone(AMH) is the only factor that can prefectly inhibit the initial follicle recruitment and FSH-stimulated follicle growth,however,stem cell factor(SCF) was the most important granulosa cell-derived growth factor that can directly stimulate the primordial to primary follicle transition. In the present ,The molecular mechanism that AMH regulates SCF has not been investigated in detail, we have found that serum levels of AMH and SCF showed a negative correlation in patients with PCOS and POF and AMH inhibit the production of SCF in culutured granulosa cells.In order to research the moloecular mechanisms we fristly determine AMH is a negative regulator of SCF by using gene cloning and siRNA respectively in vitro.Then we adopt technologies including Chromatin Immunoprecipitation ,EMAS and Luciferase Report Gene to elucidate the mechanisms by which AMH inhibits the expression of SCF through the cAMP signal pathway. In cultured rat ovaries ,we detect the expression of SCF and count the the number of follicles at each developmental stage in absence of AMH . Moreover we establish the clinical database to state the abnormal expression of AMH and SCF is a prominent reason for PCOS and POF. According to the above, Elucidation of the regulatory mechanisms involved in primordial follicle development will provide potential therapeutic targets to develop the new drugs and treat disease states such as PCOS and POF.

卵泡募集是影响女性生育力的关键环节，其过程受众多因子调控。抗苗勒氏管激素(AMH)是唯一抑制卵泡募集的因子,而干细胞因子(SCF)是促进募集的重要因子。我们前期研究发现：多囊卵巢综合征(PCOS)和卵巢早衰（POF）患者血清AMH和SCF显著负相关；基因重组AMH可抑制颗粒细胞SCF的分泌。目前AMH调控SCF的分子机制尚不清楚。本项目拟采用基因克隆、siRNA技术在细胞水平明确AMH对SCF的调控，以免疫共沉淀、EMSA、荧光素酶报告基因阐明AMH通过CAMP途径调控SCF的分子机制；通过在体靶向干扰大鼠卵巢中的AMH，研究其对SCF的表达、颗粒细胞功能、各级卵泡转化及大鼠生育力的影响；建立PCOS和POF临床数据库，分析AMH和SCF的异常表达与这两种疾病表型间的关系。最终探索AMH调控SCF的分子机制在抑制卵泡募集中的意义，也为研究PCOS和POF的发病机制、预防、诊治提供新的途径。

掌控卵泡募集的机制是延长女性生育力的核心，抗苗勒氏管激素（AMH）是唯一抑制卵泡募集的因子,而干细胞因子（SCF）是促进卵泡募集的重要因子，目前关于AMH调控SCF的机制尚无报道。我们前期研究发现：多囊卵巢综合征（PCOS）和卵巢早衰（POF）患者血清中AMH和SCF显著负相关。为深入研究AMH调控SCF的分子机制，本项目采用基因克隆、siRNA等技术在细胞水平确定AMH对SCF的负调控，以染色质免疫共沉淀、EMSA、荧光素酶报告基因阐明AMH通过抑制cAMP途径调控SCF的分子机制；建立PCOS和POF临床数据库，分析AMH和SCF等基因的表达与PCOS和POF两种疾病表型间的关系，探索AMH负调控SCF在抑制卵泡募集中的意义。研究结论：1)COV434经pIＲES2 -EGFP -AMH干预后，SCFmRNA及蛋白的表达显著减少。2)COV434经AMHsiRNA-4干预后,SCFmRNA及蛋白的表达显著增多。3)COV434经干预AMH基因表达后,VEGF/TNF-a/ BMP-15/GDF-9mRNA表达无差异。4)AMH通过cAMP/PKA途径负调控人卵巢黄素化颗粒细胞SCF的表达。5)AMH及AMH II基因的多态性可能与卵巢储备异常相关。6）PCOS患者的BMI、LH、T水平与AMH II 基因的多态性相关。7）卵泡液、黄素化颗粒细胞中AMH与SCFmRNA、蛋白质存在明显负相关。通过阐明AMH负调控SCF具体分子机制及综合分析AMH和SCF与PCOS、POF患者表型间的关系最终阐明AMH负调控SCF分子机制在抑制卵泡募集中的作用途径，试图探索PCOS和POF的发病机制，同时为PCOS和POF的预防，诊断，新药的研制提供理论依据。