【作者】 马丽；

【导师】 曹霖； 朱焰；

【作者基本信息】 复旦大学 ， 药理学， 2008， 博士

【摘要】 目的:建立子宫内膜异位症动物模型和大鼠原代培养垂体细胞模型,探讨诺美孕酮对子宫内膜异位症的药效作用和作用机制。方法:1.体外释放实验测定诺美孕酮微球的体外释放曲线:选用动情周期规则大鼠,单次皮下注射诺美孕酮微球,阴道涂片,观察诺美孕酮对SD大鼠性周期的影响,确定诺美孕酮微球是否具有长效作用。2.利用ICR小鼠,以皮下注射法建立一种新的皮下子宫内膜异位症模型,并做组织学观察和评价。3.①考察诺美孕酮微球对子宫内膜异位症（EMs）大鼠的药效作用:外科移植法建立大鼠EMs模型,三周后剖腹,模型成功者随机分为五组,分别为模型对照组,R2323（1mg/kg.14d）组,诺美孕酮微球0.5mg/kg.d、2.0mg/kg.d和4.0mg/kg.d组。给药3周后解剖,测量异位内膜体积,了解诺美孕酮微球对大鼠异位内膜体积的影响,解剖时,腹主动脉取血,磁分离酶联免疫测定法测量大鼠血清雌二醇（E₂）、孕酮（P）水平;记录子宫卵巢重量,计算脏器系数;子宫组织切片,HE染色,测量子宫内膜上皮高度。②考察诺美孕酮微球对EMs小鼠的药效作用:皮下注射法建立小鼠EMs模型,成功者随机分为七组,分别为正常组、模型对照组,亮丙瑞林（0.02mg/kg.d）组,诺美孕酮微球3、6、12和24.0mg/kg.d组,给药后3周后解剖,测量异位内膜体积,了解诺美孕酮微球对大鼠异位内膜体积的影响;解剖时,腹主动脉取血,ELISA法测度小鼠血清促卵泡激素（FSH）和促黄体激素（LH）水平;记录子宫、卵巢重量,计算脏器系数;子宫组织切片,HE染色,测量子宫内膜上皮高度。4.①解剖同时,取出小鼠子宫、异位内膜、脑和垂体,固定、切片,免疫组化ABC法测定各组动物子宫及移植异位内膜的雌激素受体（ERα）和孕激素受体（PR）的表达、下丘脑ERα、ERβ和PR的表达、下丘脑弓状核和垂体Kiss-1及其受体GPR54的表达,考察诺美孕酮对小鼠ER、PR、Kiss-1和GPR54表达的影响。②利用大鼠腺垂体组织,采用胰酶消化,体外培养大鼠原代垂体细胞,培养72小时后,加入不同浓度诺美孕酮培养49小时,分别采用GnRH、蛋白激酶C激活剂（PMA）和高浓度钾离子激发垂体细胞的促卵泡素（FSH）和促黄体素（LH）的分泌1小时,考察蛋白激酶C和电压依赖性钙离子通道,在诺美孕酮对垂体细胞分泌FSH和LH的影响中的作用。③解剖同时,迅速取出小鼠脑组织,保存于-70℃液氮,采用Biospring信号转导抗体芯片,比较模型组小鼠和诺美孕酮12mg/kg.d剂量组小鼠下丘脑蛋白表达差异,考察诺美孕酮对下丘脑信号转导通路抗体芯片中蛋白表达的影响。结果:1.体外释放试验显示,在体外,一个月的微球经28天可累积释放约85.1%,每天释放约3.04%:三个月的微球经91天可累积释放约96.5%,每天释放约1.06%。SD大鼠单次皮下注射诺美孕酮微球20070109A或20070109B（80mg/kg.3m）后,动情间期维持时间分别为77.00±1.26天和79.67±4.13天,动情间期延长。2.小鼠造模成功率为83.33%,异位灶主要位于皮下肌层,少数位于游离的皮下黏膜,呈透明的囊状,囊内有清亮的液体积聚;镜下可见囊内壁有内膜上皮细胞生长。3.①大鼠模型造模成功率为86.9%,诺美孕酮2和4mg/kg.d能显著降低异位内膜体积,抑制率显著高于模型组;诺美孕酮0.5、2和4mg/kg.d能显著降低子宫和卵巢脏器系数;诺美孕酮0.5和2mg/kg.d能显著降低子宫内膜上皮层高度;诺美孕酮0.5和2mg/kg.d能显著降低血清E₂水平;对血清P水平无影响。②小鼠模型造模成功率为82.5%,诺美孕酮6、12和24mg/kg.d能显著降低异位内膜体积,抑制率显著高于模型组;诺美孕酮3、6、12和24mg/kg.d能显著降低卵巢脏器系数,对子宫脏器系数无影响;诺美孕酮对小鼠子宫内膜上皮层高度无影响:诺美孕酮6和12mg/kg.d能显著降低血清FSH和LH水平。4.①异位内膜上表达的ERα显著低于子宫内膜,PR无显著差异;诺美孕酮3、6、12和24mg/kg.d能显著降低子宫内膜ERα和PR的表达;诺美孕酮6、12和24mg/kg.d能显著降低下丘脑ERα的表达,6mg/kg.d能显著升高ERβ的表达,诺美孕酮各剂量组均显示ERα/ERβ比值显著下降;诺美孕酮6、12和24mg/kg.d能显著降低下丘脑PR的表达。诺美孕酮3、6、12和24mg/kg.d能显著降低下丘脑ARC区Kiss-1和GPR54的表达;诺美孕酮3、6和12mg/kg.d能显著降低腺垂体Kiss-1和GPR54的表达,24mg/kg.d仅显著下调腺垂体Kiss-1的表达。②与对照组相比,10^-5和10^-6mol.L^-1诺美孕酮能显著抑制GnRH诱导的腺垂体细胞LH释放,10^-5、10^-5、10^-8和10^-10mol.L^-1诺美孕酮能显著抑制GnRH诱导的FSH释放;10^-5、10^-6、10^-8和10¹⁰mol.L^-1诺美孕酮能显著抑制PMA诱导的LH释放,10^-5、10^-6和10^-10mol.L^-1诺美孕酮能显著抑制PMA诱导的FSH释放:10^-5、10^-6和10^-8mol.L^-1诺美孕酮能显著抑制高钾离子诱导的LH释放,10^-5和10^-6mol.L^-1诺美孕酮能显著抑制高钾离子诱导的FSH释放。③下丘脑的Biospring信号转导抗体芯片结果显示,与模型对照组相比,在164个测定的目标蛋白中,诺美孕酮使48个蛋白表达发生差异,其中显著上调蛋白仅有2个:抗肌萎缩蛋白和碱性磷酸酶（AP）;而诺美孕酮显著下调蛋白质有46个:主要包括（1）激素相关通路、（2）细胞因子相关通路（3）血管发生相关通路、（4）致癌基因相关通路、（5）细胞凋亡相关通路、（6）其他:细胞周期、神经细胞相关、腺病毒相关、肿瘤标记物及个别蛋白。结论:诺美孕酮微球体内外释放稳定,具有长效缓释作用。ICR小鼠皮下子宫内膜异位症模型,简单易行,稳定可靠,为成功的Ems新动物模型。诺美孕酮微球通过抑制血清E₂、FSH和LH水平,降低实验动物的子宫和卵巢脏器系数,降低子宫内膜层高度,抑制异位内膜的生长,发挥对EMs大鼠和小鼠模型的治疗作用。首次发现,诺美孕酮能选择性降低子宫的ERα和PR的表达;诺美孕酮能作用于HPOA轴的上位,在下丘脑,诺美孕酮选择性降低ERα和PR的表达,一定程度上促进ERβ的表达,诺美孕酮对子宫和下丘脑ERα、ERβ和PR的选择性调节作用,与诺美孕酮治疗子宫内膜异位症的作用机制有关。本研究首次发现,与正常组相比,模型组小鼠下丘脑ARC区Kiss-1和GPR54表达显著升高,而模型组小鼠Kiss-1/GPR54的过度表达,可能与EMs的发病机制相关。诺美孕酮作用于ARC发挥激素负反馈作用,反馈性下调ARC区Kiss-1和GPR54的表达。诺美孕酮对Kiss-1-GPR54-GnRH-GnRH受体-促性腺激素分泌级联通路的抑制作用,与诺美孕酮治疗子宫内膜异位症的作用机制有关。首次发现,诺美孕酮不仅下调Kiss-1/GPR54在腺垂体的表达,还抑制GnRH诱导的垂体细胞FSH和LH的分泌,这种抑制作用与GnRH信号通路上的蛋白激酶C和电压依赖性钙离子通道有关。抗体芯片发现,诺美孕酮对GnRH通路上的Raf-1表达也有抑制作用,进一步说明诺美孕酮对GnRH信号通路上多个元件的调节作用,与诺美孕酮治疗子宫内膜异位症的作用机制有关。芯片结果验证,诺美孕酮的作用虽与垂体PKC有关,但与下丘脑PKC无关;FSH-b的下调与诺美孕酮对FSH的下调相关。诺美孕酮治疗子宫内膜异位症的作用机制与雄激素受体无关,而与孕酮受体相关。芯片结果首次发现:①诺美孕酮通过抑制IL-6、HGF和NF kappaB等一系列细胞因子相关蛋白的表达,调控细胞因子相关通路;②诺美孕酮通过对血管发生相关蛋白表达的抑制,调控血管发生相关通路;③在下丘脑,诺美孕酮显著下调相关致癌基因的蛋白表达,调控致癌相关基因的蛋白表达相关通路;④诺美孕酮对下丘脑神经元细胞的凋亡通路主要是促进作用,也有一定的抑制作用,诺美孕酮对下丘脑神经元的细胞凋亡的具有双向调节作用,以上发现均与诺美孕酮治疗子宫内膜异位症的作用机制有关。此外,诺美孕酮治疗子宫内膜异位症的作用机制,还可能与细胞周期相关蛋白、神经细胞相关蛋白、肿瘤标记物等有关。更多还原

【Abstract】 Aim:To establish animal endometriosis model and pituitary cell model of rat primary culture,and to study the pharmacodynamic action and mechanism of action on endometriosis.Methods:1.The release curve of nomegestrol microballoons in vitro was determine by Release Experiment in vitro;The inhibition on sexual cycle and oulation on SD rat for single administration of nomegestrol microballoons was observed.2.A new ICR mouse model with subcutaneous implants was established by hypodermic injection,and then pathohistologic chanracteristics were observed under the microscope.3.①The pharmacodynamic action of nomegestrol microballoons on rats EMs model was investigated:The model of endometriosis in rats was made by surgically autotransplanting endometrium to the peritoneum.After 3 weeks,the rats were laparotomized to investigate the growth of transplantation.The rats with peritoneal implants were randomly assigned to 5 groups:model control group; R₂₃₂₃ group（1mg/kg.14d）;nomegestrol microspheres group（doses of 0.5,2,4 mg/kg.d）.After administrations for 3 weeks,the rats were laparotomized again and the volumes of peritoneal implants were mesured.When killing the rats,blood samples in abdominal aortas were collected to measure levels of serum E2,P, using enzymeimmunometric assay（EIMA）.The weight of uterus and ovary was recorded for calculating the organ module.Uterus sections were made,then pathohistologic chanracteristics were observed under the microscope.②The pharmacodynamic action of nomegestrol microballoons on mice EMs model was investigated:The model of endometriosis in mice was made by hypodermic injection.After 3 weeks,the mice were laparotomized to investigate the growth of transplantation.The mice with subcutaneous implants were randomly assigned to 7 groups:normal group,model control group;LHRH group（0.02mg/kg.d）; nomegestrol microspheres group（doses of 3,6,12,24mg/kg.d）.After administrations for 3 weeks,the mice were laparotomized again and the volumes of subcutaneous implants were mesured.When killing the mice,blood samples in abdominal aortas were collected to measure levels of serum FSH,LH,using ELISA.The weight of uterus and ovary was recorded for calculating the organ module.Uterus sections were made,then pathohistologic chanracteristics were observed under the microscope.4.①When killing the mice,IHC（Immunohistochemistry,ABC method） was used to evaluate ERαand PR expression in the uteri and explants of different groups of mice;IHC was used to evaluate ERα,ERβand PR expression in the hypothalamus of different groups of mice;IHC was used to evaluate kiss-1 and GPR54 expression in nucleus arcuatus hypothalami and pituitary of different groups of mice,to study the effect ofvomegestrol on ER、PR、kiss-1 and GPR54 expression.②Rat anterior pituitary was digested with pancreatic enzyme,rat primary pituitary cells were cultured in vitro for 72h and then treated with different concentration of nomegestrol for 49h.The FSH and LH secretion of the cells was induced by GnRH,PMA and high[K+]e for 1h,to study the action of PKC and VSCC in the effect of nomegestrol on the FSH and LH secretion of the cells.③When killing the mice,the mouse brains were gained quickly and stored in liquid nitrogen of -70℃.By the Biospring signal transduction array the difference of protein expression in hypothalamus of EMs model group and nomegestrol 12mg/kg.d group was compared,to study the effect of nomegestrol on the protein expression in Biospring signal transduction array.Results:1.Release profiles of microspheres for one month in vitro showed that accumulated release of 28 days was 85.1%,release 3.04%per day;Release profiles of microspheres for three months in vitro showed that accumulated release of 91 days was 96.5%,release 1.06%per day.Anestrum of SD rat were maintained for 77±1.26 days and 79.67±4.13 days by injecting the 20070109A or 20070109B （80mg/kg.3m）.2.Achievement ratio of the model was 83.33%,subcutaneous implants located on muscular layer subcutaneouly,some was in mucous membrane subcutaneouly, looked diaphanous capsular with fluid in it;endomembrane epithelial cell was in the inner wall of the capsular.3.①Achievement ratio of Rat EMs model was 86.9%;Both nomegestrol 2 and 4mg/kg.d decreased the volumes of ectopic endometria,suppression efficient was significant;nomegestrol 0.5,2 and 4mg/kg.d decreased the uterus and ovary organ index significantly;nomegestrol 0.5 and 2mg/kg.d decreased the height of endometrium epithelium and serum E2 level significantly;nomegestrol had no effect on serum P level.②Achievement ratio of mouse EMs model was 82.5%;nomegestrol 6,12 and 24mg/kg.d decreased the volumes of ectopic endometria,suppression efficient was significant;nomegestrol 3,6,12 and 24mg/kg.d decreased the ovary organ index significantly,but not the uterus organ index;nomegestrol had no effect on the height of endometrium epithelium;and serum E₂ level significantly; nomegestrol had no effect on serum P level,nomegestrol 6 and 12 mg/kg.d decreased t serum FSH and LH level significantly.4.①ERαexpression in ectopic endometria was lower than in eutopic endometria, but PR expression had no difference between ectopic endometria and eutopic endometria;nomegestrol 3,6,12 and 24mg/kg.d decreased ERαand PR expression in eutopic endometria.In hypothalamus,nomegestrol 6,12 and 24mg/kg.d selectively decreased ERαand PR expression,all of nomegestrol dose group decreased the ERα/ERβratio,and nomegestrol 6mg/kg.d increased ERβexpression.In nucleus arcuatus hypothalam（ARC）, nomegestrol 3,6,12 and 24mg/kg.d decreased Kiss-1 and GPR54 expression significantly.In anterior pituitary,nomegestrol 3,6 and 12mg/kg.d decreased Kiss-1 and GPR54 expression significantly;nomegestrol 24mg/kg.d only decreased Kiss-1 expression significantly.②Compared with the control cells without drug treatment,in pituitary primary culture cells,10^-5 and 10^-6mol.L-1 nomegestrol inhibited GnRH induced LH release,10^-5、10^-6、10^-8 and 10^-10mol.L^-1 nomegestrol inhibited GnRH induced FSH release,10^-5、10^-6、10^-8 and 10^-10mol.L^-1 nomegestrol inhibited PMA induced LH release,10^-5、10^-6、and 10^-10 nomegestrol inhibited PMA induced FSH release,10^-5、10^-6 and 10^-8mol.L^-1 nomegestrol inhibited high[K+]e induced LH release,10^-5 and 10^-6 mol.L^-1 nomegestrol inhibited high[K+]e induced FSH release.③Biospring signal transduction array data of hypothalamic showed that, compared with model control group,of the 164 target proteins,nomegestrol 12mg/kg.d up-regulated two proteins and down-regulated 46 proteins.The two up-regulated proteins included Dystrophin and Alkaline Phosphatase.The 46 down-regulated proteins included（1） hormonal related protein,（2） cytokine related protein,（3） angiogenesis related protein,（4） oncogene related protein, （5） apoptosis related protein,（6） others:Cell cycle related protein,neuroscience related protein,adenovirus related protein,tumor marker related protein and some individual proteins.Conclusions:Nomegestrol microspheres delayed release in SD rat.ICR mouse subcutaneous EMs model was simply established and stable,was a successful animal EMs model.Nomegestrol microspheres inhibited ectopic endometria in EMs rat and mouse model by decreasing serum E₂,FSH and LH level,decreasing the height of endometrium epithelium,decreasing the uterus and ovary organ index.ERαand PR expression in eutopic endometria was decreased selectively in nomegestrol treated group;nomegestrol could effected on the top section-hypothalamus and pituitary;in hypothalamus,nomegestrol selectively decreased ERαand PR expression,some extent increased ERβexpression,the selective regulation of nomegestrol on ERα,ERβand PR expression related to the mechanism of nomegestrol for its action on Endometriosis,which we first found.Compared with normal group,we first found the Kiss-1 and GPR54 expression in nucleus arcuatus hypothalam（ARC） of mouse EMs model was increased significantly,which related to the pathogenesy of EMs;Nomegestrol could effect on ARC by negative feedback,decreasd Kiss-1 and GPR54 expression in ARC; Inhibition of nomegestrol on Kiss-1-GPR54-GnRH-GnRH receptor-FSH/LH secrtion cascade pathway related to the mechanism of nomegestrol for its action on Endometriosis,which we first found.Nomegestrol decreased not only Kiss-1 and GPR54 expression in pituitary,but also GnRH induced FSH and LH scretion in rat primary pituitary culture cells, dependence of PKC and VCSS,which related to the mechanism of nomegestrol for its action on Endometriosis. Data in array analysis verificated that effects of nomegestrol on pituitary was PKC-related,but not on hypothalamus.The downregulation of Nomegestrol on FSH-b related to the inhibition of Nomegestrol on serum FSH level.PR expression difference related to the mechanism of nomegestrol for its action on Endometriosis, but not androgen receptor.Data in array analysis firstly found that:①Nomegestrol regulated cytokine correlation pathway by inhibiting cytokine related proteins expression such as IL-6, HGF,NF kappa B,which related to the mechanism of nomegestrol for its action on Endometriosis.②Nomegestrol regulated angiogenesis correlation pathway by inhibiting angiogenesis related proteins expression,which related to the mechanism of nomegestrol for its action on Endometriosis.③Nomegestrol regulated oncogene correlation pathway by inhibiting oncogene related proteins expression,which related to the mechanism of nomegestrol for its action on Endometriosis.④Nomegestrol two-way regulated apoptosis correlation pathway by inhibiting or promoting apoptosis related proteins expression,which related to the mechanism of nomegestrol for its action on Endometriosis.Beside these,cell cycle related protein,neuroscience related protein,adenovirus related protein,and tumor marker related protein perhaps related to the mechanism of nomegestrol for its action on Endometriosis.更多还原