【作者】 杨静；

【导师】 黄荷凤；

【作者基本信息】 浙江大学 ， 妇产科学， 2015， 博士

【摘要】 第一部绝经后妇女血清FSH与儿茶酚胺和血压水平相关性目的：了解绝经后妇女血清FSH水平与儿茶酚胺水平相关性。材料和方法：收集来我院进行体检,没有基础疾病及内分泌疾病,过去三个月没用过激素类药物治疗的绝经后妇女,严格按照最新妇女生育老化STRAW+10进行分级,将她们分为Stage+1a+1b, Stage+1c绝经后及育龄期Stage-4,-3b三组。记录她们的身高、体重、腰臀比、血压等指标,利用HPLC-ECL法检测血清中的儿茶酚胺的水平,用免疫学方法检测血清中的性激素六项,其血脂水平来自于浙江大学医学院附属妇产科医院化验室检测结果。结果：1.绝经后血清中儿茶酚胺水平增加,与血清FSH和E2水平相关。2.绝经后妇女Stage+1a+1b血清儿茶酚胺水平升高最明显,此时期血压升高也最明显。3.绝经后妇女血清中儿茶酚胺的浓度与FSH的浓度成中度正相关。结论：绝经后妇女血清中儿茶酚胺,尤其是去甲肾上腺素水平与血清FSH水平正相关。第二部分大鼠动物模型血清FSH水平与儿茶酚胺和血压的关系目的：了解去势动物血清FSH水平与儿茶酚胺水平的关系。材料和方法：应用SD大鼠分别建立四种动物模型：假手术组、卵巢去势组、肾上腺切除+皮质酮及醛固酮渗透泵植入组、卵巢去势+肾上腺切除+皮质酮及醛固酮渗透泵植入组及卵巢去势+GnRHa注射组,并注射人重组卵泡刺激素和或17β雌二醇。注射后14天后检测动物收缩压、舒张压、平均动脉压及心率、血清性激素含量及血清儿茶酚胺水平。5个月后行肠系膜张力试验,观察小动脉收缩功能。结果：发现：1.卵巢去势大鼠较假手术组血清FSH水平及血清儿茶酚胺水平均明显升高,血压水平也呈增高趋势。2.卵巢去势+肾上腺切除+皮质酮及醛固酮渗透泵植入组大鼠血清FSH水平升高,血清儿茶酚胺水平升高不明显,血压水平也无明显增高趋势。3.卵巢去势+GnRHa注射组大鼠血清FSH水平及血清儿茶酚胺水平均无明显升高,血压水平也无明显增高趋势。4.卵巢去势大鼠注射雌激素不能完全纠正血清FSH水平及血清儿茶酚胺水平升高,也不能完全纠正升高的血压。5、卵巢去势+GnRHa注射组大鼠注射外源FSH后,血清儿茶酚胺水平及血压水平均明显升高。4.大鼠肠系膜功能实验表明：不论补充雌激素与否,血清FSH水平高大鼠其肠系膜收缩功能均明显降低。结论：卵巢去势大鼠血压升高与血清儿茶酚胺水平升高有关,肾上腺髓质依赖性的儿茶酚胺水平升高与血清FSH水平升高有关。第三部分FSH促进肾上腺髓质细胞合成儿茶酚胺的细胞内机制目的：证实FSHR在肾上腺髓质嗜铬细胞表达,FSH可以通过与FSHR结合促进儿茶酚胺合成。探讨FSH促进肾上腺髓质嗜铬细胞合成和分泌儿茶酚胺的细胞内机制。材料和方法：应用RT-PCR、western blot及免疫荧光技术检测FSH受体在大鼠肾上腺嗜铬瘤细胞系(PC12)、大鼠肾上腺髓质嗜铬细胞及人肾上腺髓质嗜铬细胞膜上表达。应用反相高效液相电化学法及活细胞工作站实时图像分析法检测FSH对PC12细胞及大鼠肾上腺原代髓质嗜铬细胞合成儿茶酚胺的影响。应用FSHR特异的siRNA转染的方法建立细胞体外敲除模型。应用腺病毒转染技术构建FSHR高表达细胞模型。用western-blot方法及免疫荧光技术对FSH促进儿茶酚胺合成有关的激酶活性进行检测。结果：1.FSH受体在大鼠肾上腺嗜铬细胞瘤细胞、大鼠肾上腺髓质嗜铬瘤细胞及人肾上腺髓质嗜铬细胞表达。2.FSH可以促进PC12细胞及大鼠肾上腺髓质原代嗜铬细胞合成去甲肾上腺素和或肾上腺素。3.FSH作用于FSHR通过Gsa激活PKA、ERK1/2、JNK使得下游信号分子磷酸化,主要通过激活p-CREB、p-c-jun、 p-ERK及p-AKT调节儿茶酚胺的合成。结论：FSHR在肾上腺髓质嗜铬细胞瘤表达。FSH通过激活FSHR促进PC12细胞及大鼠肾上腺髓质原代嗜铬细胞合成去甲肾上腺素和或肾上腺素。FSH主要通过激活p-CREB、p-c-jun、p-ERK及p-AKT通路调节儿茶酚胺的合成。更多还原

【Abstract】 Part Ⅰ Correlation between FSH and catecholamine in the serum of postmenopausal women.Objective:To investigate the correlation between FSH and catecholamine in the serum of postmenopausal women.Materials and methods:To collect the data of women who came to Women Hospital, School of Medicine, Zhejiang University for physical examination, who had no basic diseases such as diabetes or cardiovascular diseases and taken hormone-like medicine. Their data included physique data, sex hormone, Blood lipid level, blood pressure, serum catecholamine. According to the latest women aging STRAW+10, we collected the data of three groups such as Stage+1a+1b, Stage+1c and Stage-4,-3b and analyzed their relationships between them ultimately to find the reason for increasing blood pressure in post-menopause. The specific methods as followed:reverse high performance liquid chromatography-electrochemical detector was used to detect the concentration of serum catecholamine. Immunological methods were used to detect the concentration of serum sex hormones. The blood lipid levels and height, weight, waist-hip ratio, blood pressure and heart rate were from the Laboratory Medicine from Women Hospital. The data was analyzed by SPSS19.0.It is statistically significant when P is less than0.05.Results:1. The concentration of serum catecholamine are elevated in post menopause, which are associated with serum follicle-Stimulating Hormone (FSH) and estrogen, especially with FSH elevation.2. The elevation of serum catecholamine is the most obvious during times of sex hormones volatility-Stage+la+lb, Simultaneously the elevation of blood pressure is the highest.3. The concentration of serum catecholamine is mildly associated with the concentration of serum FSH.Conclusions:Postmenopausal hypertension is due to the elevation of serum catecholamine, especially norepinephrine. The elevation of serum catecholamine is associated with estrogen loss and FSH elevation, especially the later. Part Ⅱ Corelation of follicle stimulating hormone (FSH),catecholamine and blood pressure (BP) in SD female ratsObjective:Using animal models to investigate the correlation between FSH and NE, EPI and BP.Materials and methods:Four animal models (ovariectomized, adrenalectomized, ovariectomized and adrenalectomized, and gonadotropin-releasing hormone agonist injected and ovariectomized models) were used. We designed four animal models. The first one is separated into the sham groups(S1) and bilateral ovariectomized groups; the second one included the sham groups (S2) and bilateral adrenalectomized groups implanted into physical concentration of corticosterone and aldosterone osmotic pumps; the third one is the sham groups (S3) and bilateral ovariectomized and adrenalectomized groups implanted into physical concentration of corticosterone and aldosterone osmotic pumps; the fourth group is the sham groups (S4) and bilateral ovariectomized groups injected into gonadotropin-releasing hormone agonist. Human recombinant follicle-stimulating hormone and/or estrogen were successively injected in different groups to evaluate the effect of FSH and estrogen. The blood pressure, sex hormone and serum catecholamine of All Sprague Dawley (SD) rats were measured in14days after the operation and injection. In5months after operation and injection, the mesenteric tension experiment was done for investigating the changes the systolic function of small mesenteric artery.Results:1. The postmenopausal hypertension model of SD rats models is successful. Postmenopausal hypertension of rats is relied on the existence of bilateral adrenals.2. Postmenopausal hypertension of SD rats was jointly caused by estrogen loss and follicle-stimulating hormone elevation. After bilateral ovariectomy, even estrogen is enough supplied, postmenopausal hypertension can not be completely rectified, which showed that elevated FSH is involved into regulating blood pressure in addition of estrogen loss.3. The variation tendency of FSH in SD rats was consistent with the tendency of norepinephrine and epinephrine, which showed the association of FSH and norepinephrine/epinephrine existed. In other words, when serum FSH elevated, the levels of norepinehprine/epinephrine increased, vice versa.4. The experiment of mesenteric artery function in rats showed that:whether estrogen was replaced or not, the elastical function of mesenteric artery in SD rats with high levels of serum FSH decreased.Conclusions:The four SD models demonstrated that postmenopausal hypertension is relied on the adrenal medulla. Postmenopausal hypertension is due to the elevation of catecholamine and the decrease of elastic function of small artery that caused by norepinehprine elevation. The catecholamine increase is associated with serum FSH. Part Ⅲ The intracellular mechanism of FSH promoting catecholamine synthesis in PC12and rat adrenal medulla cellsObjective:To explore the mechanism of FSH involved catecholamine synthesis in PC12cells and rat primary adrenal medulla cellse.Materials and methods:1.The expression profile of FSHR was investigated in PC12cells, rat primary adrenal medulla chromatin cells by RT-PCR, western blot and immunofluorescence staining. The effect of FSHR on the regulation of NE synthesis was evaluated by Reverse phase high performance liquid chromagraphy electrochemical technology in PC12cells and rat primary adrenal medulla chromatin cells. We explored the intracellular mechanism of FSHR involved in the NE synthesis of PC12cells in vitro.Results:1. The expression profile of FSHR was investigated in PC12cells, rat primary adrenal medulla chromatin cells.2. Different concentration of human recombinant FSH can promote the synthesis of norepinephrine and epinephrine of PC12cells and rat adrenal medulla chromaffin cells.3.FSH combined with FSHR stimulated Gsα, then activated PKA, ERK1/2, JNK ultimatedly phosphorylated CREB, c-jun, ERK and AKT that acted on response element of yrosine hydroxylase promoter to up-regulate synthesis of tyrosine hydroxylase--the rate-limiting enzyme of catecholamine synthesis, which resulted in increased synthesis of catecholamine.4. The study provides a new mechanism of postmenopausal hypertension. That is to say, elevated FSH in postmenopausal women promoted the synthesis of catecholamine by adrenal medulla chromaffin cells.Conclusions:FSH enhances tyrosine hydroxylase (TH) expression and catecholamine synthesis. FSH enhances tyrosine hydroxylase (TH) expression and catecholamine synthesis. FSHR signals activate CREB, Erk, AKT and NFκB pathways via Gsα in catecholamine synthesis.更多还原