【作者】 庄亚玲；

【导师】 黄丽丽；

【作者基本信息】 浙江大学 ， 妇产科学， 2010， 博士

【摘要】 第一部分药物流产后绒毛组织中P-糖蛋白与子宫异常出血的关系目的：通过检测P-糖蛋白在药流成功组和出血组绒毛组织中的表达,以及检测在人早孕期滋养细胞内米非司酮的浓度,探讨P-糖蛋白与药物流产后子宫异常出血的关系及其可能机制。材料与方法：选择自愿要求行药物流产的早孕妇女,收集服米索前列醇当天排出的绒毛组织,根据随访结果分药流成功组(37例)和出血组(37例)。采用免疫组织化学法和蛋白免疫印迹(Western Blotting)检测两组绒毛组织中P-糖蛋白的表达水平。体外培养早孕期绒毛滋养细胞,分实验组(20μM CsA组和40μM CsA组)和对照组,以及每组加米非司酮(分0.5μM组、1.25μM组和2.5μM组)后,用高效液相法分析绒毛滋养细胞内米非司酮的浓度。结果：P-糖蛋白主要定位于合体滋养细胞膜上；药流出血组P-糖蛋白的表达水平高于药流成功组(P<0.05)；不同米非司酮浓度(0.5μM、1.25μM和2.5μM)的各组(20μM CsA组、40μM CsA组和对照组)间均无显著性差异(P>0.05)。结论：药物流产后绒毛组织中P-糖蛋白的高表达可能与子宫异常出血有关；在滋养细胞,P-糖蛋白不是通过将米非司酮从细胞内泵出,降低细胞内的浓度起作用的,即米非司酮可能不是P-糖蛋白的作用底物。第二部分药物流产后绒毛组织中MMP-2、MMP-9和EMMPRIN与子宫异常出血的关系目的：通过检测绒毛组织中MMP-2和MMP-9活性和其蛋白以及EMMPRIN在药流成功组和出血组的表达,探讨EMMPRIN、MMP-2和MMP-9与药物流产后子宫异常出血的关系。材料与方法：选择自愿要求行药物流产的早孕妇女,收集服米索前列醇当天排出的绒毛组织,根据随访结果分药流成功组(37例)和出血组(37例)。采用明胶酶谱法检测绒毛组织中MMP-2和MMP-9的活性,采用免疫组织化学法检测两组绒毛组织中MMP-2和MMP-9的蛋白表达水平。采用免疫组织化学法和蛋白免疫印迹(Western Blotting)检测两组绒毛组织中EMMPRIN蛋白表达水平。结果：绒毛组织中MMP-9表达水平药流出血组高于药流成功组(P<0.001),而MMP-2的表达水平两组无显著性差异(P>0.05)。两组绒毛组织中均未检测到MMP-2和MMP-9的活性。EMMPRIN主要定位于合体滋养细胞和细胞滋养细胞胞浆和胞膜,蛋白表达水平药流出血组低于药流成功组(P<0.05)。结论：药物流产后绒毛组织中MMP-9高表达及EMMPRIN低表达可能与子宫异常出血有关；而MMP-2表达可能与子宫异常出血无关。第三部分人滋养细胞P-糖蛋白与EMMPRIN的相互作用目的：探讨P-糖蛋白和EMMPRIN之间的相互作用。材料与方法：选择自愿要求行人工流产术的早孕妇女,收集行人工流产时的绒毛组织进行组织块培养24小时和48小时,加P-糖蛋白抑制剂(分20μM CsA组和40μM CsA组),采用Western Blotting法检测EMMPRIN蛋白的表达水平；加EMMPRIN单克隆抗体(分2.5μg／ml MEM-M6／1组和5.0μg／ml MEM-M6／1组),采用Western Blotting法检测P-糖蛋白的表达水平。结果：加P-糖蛋白抑制剂24小时和48小时后,EMMPRIN的表达水平在20μMCsA组、40μM CsA组和对照组三者之间均无显著性差异(P>0.05)。加EMMPRIN单克隆抗体培养24小时后,P-糖蛋白蛋白水平在2.5μg／ml MEM-M6／1组、5.0μg／mlMEM-M6／1组均比对照组低,差异均有显著性(P<0.001)。培养48小时后,P-糖蛋白表达水平在2.5μg／ml MEM-M6／1组、5.0μg／ml MEM-M6／1组和对照组三者之间差异均无显著性(P>0.05)。结论：在人滋养细胞P-糖蛋白对EMMPRIN的表达可能没有调控作用,而EMMPRIN可能调控P-糖蛋白的表达。更多还原

【Abstract】 Part I The relationship of P-glycoprotein in villi and abnormal uterine bleeding after medical abortion with mifepristone and misoprostolObjectives:To investigate the relationship of P-glycoprotein and abnormal uterine bleeding after medical abortion by analyzed the expression level of P-glycoprotein in the villi, and the concentration of mifepristone in trophoblast cells of early pregnancy in vitro.Materials and Methods:Women who voluntarily wanted to terminate early pregnancy by medical abortion with mifepristone and misoprostol were recruited. The villi discharged naturally on the day using misoprostol were collected. According to the follow-up, women were divided into two groups which were completed abortion group (37 cases) and bleeding group (37 cases). The location of P-glycoprotein in the villi were examined by immunohistochemistry. The expression levels of P-glycoprotein in the villi of two groups were tested by Western Blotting. In vitro, trophoblasts were cultured with CsA (20μM and 40μM) and mifepristone (0.5μM,1.25μM and 2.5μM). The concentration of mifeprsitone in trophoblasts was detected by HPLC.Results:P-glycoprotein was located in the membrance of syncytiotrophoblast. The expression level of P-glycoprotein in bleeding group was higher than that in completed abortion group(p<0.05). There were no significant differences among three groups (20μM CsA group,40μM CsA group and Control group) in every concentration of mifepristone (0.5μM,1.25μM and 2.5μM) (p>0.05).Conclusions:The increased expression level of P-glycoprotein in the villi may be related to abnormal uterine bleeding after medical abortion. In vitro, P-glycoprotein did not pump the mifepristone out of the trophoblast to reduce the concentration of mifepristone in trophoblasts, namely mifepristone may be not the substrate of P-glycoprotein.Part II The effect of MMP-2, MMP-9 and EMMPRIN in villi on abnormal uterine bleeding after medical abortion with mifepristone and misoprostolObjectives:To investigate the effect of MMP-2, MMP-9 and EMMPRIN on abnormal uterine bleeding after medical abortion with mifepristone and misoprostolMaterials and Methods:Women who voluntarily wanted to terminate early pregnancy by medical abortion with mifepristone and misoprostol were recruited. The villi discharged naturally on the day using misoprostol were collected. According to the follow-up, women were divided into two groups which were completed abortion group (37 cases) and bleeding group (37 cases). The expression levels of MMP-2 and MMP-9 proteins were examined by immunohistochemistry. The activities of MMP-2 and MMP-9 were detected by gelatin zymography. The location of EMMPRIN in the villi were examined by immunohistochemistry. The expression levels of EMMPRIN in the villi of two groups were tested by Western Blotting.Results:There was no significant difference of MMP-2 protein expression levels in two groups (p>0.05); the expression level of MMP-9 protein in bleeding group was higher than that in completed abortion group (p<0.001). The activities of MMP-2 and MMP-9 in two groups were not detected. The expression of EMMPRIN was located in the membrance of syncytiotrophoblast and cytotrophoblast. The expression level of EMMPRIN in bleeding group was lower than that in completed abortion group (p＜0.05).Conclusions:The decreased expression level of EMMPRIN protein and the increased expression level of MMP-9 in the villi may be related to abnormal uterine bleeding after medical abortion; but the expression level of MMP-2 in the villi may be not related to abnormal uterine bleeding after medical abortion.PartⅢThe interaction of P-glycoprotein and EMMPRIN in trophoblasts of early pregnancy in vitroObjectives:To investigate the interaction of P-glycoprotein and EMMPRIN in trophoblasts of early pregnancy in vitro.Materials and Methods Women who voluntarily wanted to terminate early pregnancy by surgical abortion were recruited. The villi were collected and cultured in trophoblast explant. Under the presence of CsA (20μM group and 40μM group), trophoblast explant was cultured for 24 hours and 48 hours, the expression level of EMMPRIN protein was analyzed by Western Blotting. Under the presence of MEM-M6/1 (2.5μg/ml group and 5.0μg/ml group), trophoblast explant was cultured for 24 hours and 48 hours, the expression level of P-glycoprotein protein was analyzed by Western Blotting.Results:Under the presence of CsA (20μM CsA group and 40μM CsA group), cultured for 24 hours or 48 hours, there were no significant differences of the expression levels of EMMPRIN protein among 20μM CsA group,40μM CsA group and control group(p>0.05). Under the presence of MEM-M6/1 (2.5μg/ml MEM-M6/1 group and 5.0μg/ml MEM-M6/1 group), the expression levels of P-glycoprotein protein in two concentrations of MEM-M6/1 groups were lower than that in control group at 24 hours’ culture. There were significant differences among three groups(2.5μg/ml MEM-M6/1 group,5.0μg/ml MEM-M6/1 group and control group) (p<0.05); there were no significant differences among three groups at 48 hours’culture(p>0.05).Conclusions:In vitro, EMMPRIN protein maybe regulate the expression level of P-glycoprotein protein, but P-glycoprotein protein maybe not regulate the expression level of EMMPRIN protein.更多还原