补肾调泡周期疗法对PCO大鼠AMH/Smad信号通路影响的研究

Follicular dysplasia is the most difficult problem of polycystic ovary syndrome,which seriously affects the reproductive health of women. Anti-müllerian hormone is the negative feedback regulation mechanism of follicular development,which has becoming one of novel targets and the breakthrough points in the diagnosis and treatment of PCOS. The cycle therapy of tonifying kidney and adjusting follicle is the effective therapeutic method of treating PCOS according to menstrual period and follicular phase. Early studies have shown that the curative effect is distinct in improving the pregnancy rate and reducing adverse reactions.The project use technology of cell culture, flow cytometry, enzyme-linked immunosorbent, Western blotting and real-time fluorescent quantitative PCR etc to do experiment in vivo and in vitro. Acting on the granulosa cells of antral follicles and PCO rats with letrozole induced.To observe the effect of the drug serum of cycle therapy of tonifying kidney and adjusting follicle on the proliferation and secretion of the granulosa cells of antral follicles and the expression of AMH and Smad4 in different time points in vitro experiment.And to observe the effect of different doses of the cycle therapy of tonifying kidney and adjusting follicle on the estrus, serum hormone, ovarian morphology and the expression of AMH/Smad signaling pathways. Count pregnant rats and the number of the bed.Through the AMH/Smad signaling pathways to discuss the mechanism of the cycle therapy of tonifying kidney and adjusting follicle acting on the promoting follcles mature and ovulation of PCOS. To provide the experimental basis for prevention and control of difficult and baffling diseases such as the reproductive dysfunction.

卵泡发育异常是多囊卵巢综合征（PCOS）的最棘手问题,严重影响女性生殖健康。抗苗勒氏管激素(AMH)相当于卵泡发育负反馈调控机制，是诊治PCOS的新靶点和突破点之一。补肾调泡周期疗法是以月经期和卵泡期分期调治PCOS的有效方法，前期研究证明提高妊娠率、降低不良反应临床疗效显著。本项目通过体内和体外实验，以大鼠窦卵泡颗粒细胞和来曲唑诱导PCO大鼠为受试对象，运用细胞培养、流式细胞术、酶联免疫、蛋白质印迹、实时荧光定量PCR等技术，体外实验观察细胞培养各时间点补肾调泡周期疗法含药血清对大鼠窦卵泡颗粒细胞增殖、分泌功能及AMH、Smad4表达影响，体内实验观察该法不同剂量组合对PCO大鼠动情周期、血清激素、卵巢形态、AMH/Smad信号通路因子表达影响，计数孕鼠和着床数。从AMH/Smad信号通路探讨补肾调泡周期疗法促PCOS卵泡发育成熟并排卵的机制。为防治生殖功能障碍等疑难病症提供实验依据。

卵泡发育异常是多囊卵巢综合征（PCOS）的最棘手问题,严重影响女性生殖健康。抗苗勒氏管激素(AMH)相当于卵泡发育负反馈调控机制，是诊治PCOS的新靶点和突破点之一。补肾调泡周期疗法是以月经期和卵泡期分期调治PCOS的有效方法，前期研究证明提高妊娠率、降低不良反应临床疗效显著。本项目采用来曲唑制作PCO大鼠动物模型，运用补肾调泡周期疗法进行干预，结果发现，补肾调泡周期疗法能有效改善PCO模型大鼠动情周期紊乱；改善PCO模型大鼠卵巢组织病理学改变；明显降低PCO模型大鼠外周血清高T、高LH水平；明显降低PCO模型大鼠治疗前后体质量增加量和卵巢系数。补肾调泡周期疗法能有效调控卵巢功能，其机制与调节内分泌激素水平，调节动情周期、改善多囊卵巢组织病理形态、降低体质量和卵巢重量有关，从而促进卵泡发育成熟和恢复排卵。补肾调泡周期疗法能明显降低PCO模型大鼠卵巢组织AMH、AMH RⅡ的基因、蛋白表达及其在卵巢窦前、小窦卵泡颗粒细胞中的蛋白分布，从而调控PCO模型大鼠卵巢组织Smad4的基因、蛋白表达及其在卵巢颗粒细胞中的蛋白分布，推测补肾调泡周期疗法可能通过AMH/Smad信号通路，影响卵泡募集和选择，促进卵泡发育成熟，改善排卵障碍，恢复卵巢功能。通过补肾调泡周期疗法对PCO模型大鼠妊娠结局影响实验，发现补肾调泡周期疗法能明显提高PCO模型大鼠胎仔数，有提高妊娠率趋势，这可能与补肾调泡周期疗法治疗排卵障碍性不孕、子宫内膜容受性低、促进胚胎着床，从而改善PCO模型大鼠生殖能力有关。并初步探索了相关药物组合的交互作用及量效关系。体外培养SD大鼠窦卵泡颗粒细胞，采用FSHR免疫组化法进行颗粒细胞鉴定。采用WB和实时荧光定量PCR 检测细胞体外培养不同时间节点补肾调泡周期疗法含药血清对窦卵泡颗粒细胞AMH/Smad信号通路关键因子AMH、AMHRⅡ、Smad4蛋白和基因表达的动态影响。本研究为妇科常见、疑难病症PCOS的防治，PCOS所致卵泡发育异常和排卵障碍性不孕等临床棘手问题的解决，提供可靠的实验依据和理论支撑。