米非司酮对子宫巨噬细胞活化的作用及机制研究

发布时间：2018-04-03 13:21

本文选题：子宫　切入点：巨噬细胞　出处：《浙江大学》2017年博士论文

【摘要】：药物避孕是妇女避孕史上的里程碑。自上世纪60年代以来,甾体激素类口服避孕药(Oral contraceptives,OCs),已在全球得到了广泛的应用。但是由于口服避孕药含有雌激素,存在导致血栓性疾病、激素依赖性肿瘤及体重增加等副作用,限制了它的使用;而单纯孕激素类避孕药又由于存在不规则阴道流血及月经周期控制欠佳等缺点,影响了它在临床上的应用。因此开发和研制新一代口服避孕药,避免和减少上述的这些不良反应迫在眉睫。米非司酮是一种具有强效的抗孕激素受体和抗糖皮质激素受体特性的合成19-去甲睾酮衍生物,自其合成以来在临床上得到广泛应用,包括妇产科学、肿瘤学、免疫学及内分泌学等。目前,米非司酮也是一种在无保护性生活之后、高效的紧急避孕方法,并且已经有研究认为其有潜力成为新型的、副作用极少的长期口服避孕药;研究已经表明米非司酮并不直接对胚胎产生不利影响,但可通过作用于内膜来防止胚胎种植而又不影响排卵,从而实现"内膜避孕"。但是,米非司酮避孕的确切作用机制仍有待进一步阐明。巨噬细胞是可塑性最强的免疫细胞,存在于所有组织中,并且可以根据它们所处微环境的变化而改变其功能。由于在人类的子宫内膜中几乎没有树突状细胞,巨噬细胞就成为了最主要的抗原呈递细胞。子宫巨噬细胞可能在胚胎成功植入所需的几个过程中发挥关键作用,包括子宫内膜的重塑、相邻免疫细胞的免疫调节、滋养细胞侵袭和胎儿-母体免疫耐受的调节等。深入认识和探索米非司酮确切的避孕机理可能为新型避孕药的开发和研制提供科学依据。第一部分子官巨噬细胞在基础状态下为M2型巨噬细胞目的:明确子宫巨噬细胞在基础状态下的活化表型。方法:从早孕期妇女外周血分离单核细胞,体外分别诱导分化成经典活化(M1)型巨噬细胞及替代活化(M2)型巨噬细胞;同时从早孕期蜕膜组织分离、培养得到高纯度的子宫巨噬细胞,在显微镜下观察子宫巨噬细胞的形态,采用流式细胞术检测子宫巨噬细胞的CD14+表面标记。采用ELISA检测子宫巨噬细胞培养的上清液中的细胞因子IL-12p70、IL-23、IL-p40及IL-10的表达水平,将其分别与M1型巨噬细胞及M2型巨噬细胞分泌的相应的细胞因子相比较。结果:在本研究中,我们采用免疫磁珠分选法成功分离到了高纯度的子宫巨噬细胞(90%以上);与M1型巨噬细胞相比,子官巨噬细胞分泌的抗炎因子IL-10水平明显更高,表达的促炎因子IL-12p70、IL-23和IL-p40水平明显低下,而子宫巨噬细胞与M2巨噬细胞表达的细胞因子相似,即高IL-10水平和低IL-12p70,IL-23和IL-p40水平。结论:子宫巨噬细胞在基础状态下呈现M2表型,这可能有利于胚胎种植及妊娠的发展。第二部分米非司酮通过糖皮质激素受体拮抗作用促进M1型巨噬细胞活化目的:明确米非司酮对子宫巨噬细胞活化的作用及途径。方法:将体外分选到的高纯度子宫巨噬细胞采用不同浓度的米非司酮、米非司酮+孕酮、米非司酮+地塞米松进行处理。采用ELISA检测米非司酮、米非司酮+孕酮、米非司酮+地塞米松对子官巨噬细胞活化的影响。采用MTT检测米非司酮对子宫巨噬细胞活力的影响。结果:与未处理的子宫巨噬细胞相比,经低剂量米非司酮处理的子宫巨噬细胞产生的IL-12p70、IL-23和IL-p40明显增加,而IL-10的表达水平却明显降低;并且与经65nmol/L米非司酮处理的子宫巨噬细胞相比,经200nmol/L米非司酮处理的子宫巨噬细胞明显产生更多的IL-12p70、IL-23和IL-12p40,而抑制IL-10产生的作用更加明显。经低剂量米非司酮处理的巨噬细胞与未经处理的巨噬细胞之间的细胞活力没有显著性差异。与经200nmol/L米非司酮处理的巨噬细胞相比,孕酮对200nmol/L米非司酮诱导巨噬细胞产生的高IL-12p70、IL-23、IL-p40表达没有影响,而且不影响200nmol/L米非司酮对巨噬细胞IL-10表达的抑制。地塞米松能明显抑制200nmol/L米非司酮诱导巨噬细胞产生的IL-12p70、IL-23和IL-12p40的表达水平,同时能逆转200nmol/L米非司酮对巨噬细胞IL-10表达的抑制。结论:米非司酮通过糖皮质激素受体拮抗作用促进子宫巨噬细胞从M2型向M1型转化,从而有可能增强Th1免疫应答而抑制Th2免疫应答,最终导致胚胎植入失败,这可能是其避孕机理之一。第三部分米非司酮通过激活TAK1促进M1型巨噬细胞活化目的:明确米非司酮对子宫巨噬细胞活化的分子机制。方法:将体外分选到的高纯度子宫巨噬细胞采用200 nmol/L米非司酮、200 nmol/L米非司酮和6umol/L地塞米松进行处理。采用蛋白免疫印迹法检测NFκβ、p38 MAPK、ERK、JNK及TAK1蛋白磷酸化水平的变化。分别采用NFκβ、p38MAPK及TAK1特异性抑制剂JSH-23、SB203580及5Z-7-oxozeaenol检测其对米非司酮介导的M1型巨噬细胞活化的影响。结果:经200nmol/L米非司酮处理的子宫巨噬细胞Iκβα的磷酸化明显增加,6umol/L地塞米松能明显抑制这一作用。与抑制米非司酮介导Iκβα磷酸化一致,地塞米松也能抑制米非司酮介导的Iκβa降解。另外,地塞米松抑制了米非司酮介导的磷酸化p65的细胞内核转位。经200nmol/L米非司酮处理的子宫巨噬细胞p38 MAPK的磷酸化明显增加,地塞米松能明显抑制这一作用。JSH-23能明显抑制米非司酮诱导的IL-12p70、IL-23和IL-12p40的表达水平,但是JSH-23并不能增加IL-10的表达水平。SB203580抑制p38MAPK激活后对米非司酮诱导的经典巨噬细胞活化没有影响。经200nmol/L米非司酮处理的子宫巨噬细胞TAK1的磷酸化明显增加,地塞米松能明显抑制这一作用。5Z-7-oxozeaenol能明显抑制米非司酮诱导的NFκβ活化,并且明显抑制IL-12p70、IL-23和IL-12p40的产生,同时能明显增加IL-10的表达水平。结论:米非司酮通过激活TAK1将子宫巨噬细胞从M2型转向M1型,可能导致胚胎种植失败,从而达到避孕的效果。
[Abstract]:Contraception is milepost of contraception of women history. Since the last century since 60s, steroid oral contraceptive (Oral, contraceptives, OCs) has been widely used in the world. But due to oral contraceptives containing estrogen, lead to thrombotic diseases, hormone dependent tumors and weight gain and other side effects limit the use of it; but only progesterone contraceptive and irregular vaginal bleeding due to the presence of defects and poor control of the menstrual cycle, the effect of its clinical application. Therefore, the development and the development of a new generation of oral contraceptives, avoid and reduce the adverse reactions of the imminent. Mifepristone is a synthesis of 19- with potent anti anti progesterone receptor and glucocorticoid receptor characteristics of nortestosterone derivatives, since its synthesis has been widely used in the hospitals, including obstetrics and Gynecology, oncology, free Epidemiology and endocrinology. At present, is also a kind of mifepristone in unprotected sex after emergency contraception effective, and has been considered to have the potential to be a new, long-term side effects of oral contraceptives is few; studies have shown that mifepristone does not directly produce adverse effects on embryo, but by acting on the endometrium to to prevent the embryo implantation without affecting ovulation, so as to realize the "endometrium contraception". However, the exact mechanism of action of mifepristone for contraception remains to be elucidated. Macrophages are the immune cells of the strongest plasticity, exists in all tissues, and can change its function according to changes in their microenvironment at. Because in the human uterus almost no endometrial dendritic cells, macrophages become the main antigen-presenting cells. Uterine macrophages may succeed in embryo implantation Play a key role in the remodeling process, including endometrial immune, immune cells adjacent regulation of trophoblast invasion and fetal maternal immune tolerance regulation. Understand and explore the exact mechanism for contraception mifepristone and provide scientific basis for the development of new contraceptives. The first molecular officer of macrophages in the basal state for M2 type macrophages Objective: to clear the uterine macrophages in basal state activation phenotype. Methods: mononuclear cells were isolated from peripheral blood of pregnant women during the first trimester, in vitro were induced to differentiate into classically activated macrophages (M1) and alternative activation (M2) macrophages isolated from human decidua; at the same time, training high the purity of uterine macrophages, observe uterine macrophages morphology under the microscope, the surface markers of CD14+ detection of uterine macrophages by flow cytometry Using cytokine IL-12p70, ELISA detection of uterine macrophages supernatant in cultured IL-23, the expression level of IL-p40 and IL-10, the corresponding cytokines respectively with M1 macrophages and M2 macrophages secretion compared. Results: in this study, we use Macs successfully isolated with high purity uterine macrophages (more than 90%); compared with the M1 type macrophages, macrophage sub officer anti-inflammatory factor IL-10 levels were significantly higher, the expression of proinflammatory cytokines IL-12p70, IL-23 and IL-p40 levels were low, while cytokines uterine macrophages and M2 macrophages expressed similar, high IL-10 level and low IL-12p70, IL-23 and IL-p40 level. Conclusion: Uterine macrophages showed M2 phenotype in basal condition, it may be beneficial to the development of embryo implantation and pregnancy. In the second part, through the glucocorticoid mifepristone Type M1 receptor antagonism promotes macrophage activation Objective: effect and clear way of mifepristone on uterine macrophage activation. Methods: high purity separation to the uterine macrophages in vitro with different concentrations of mifepristone and mifepristone + progesterone and mifepristone + dexamethasone treatment. ELISA was used to detect the effect of mifepristone and mifepristone + progesterone and mifepristone + dexamethasone activation on the official macrophages. With MTT detection of mifepristone on uterine macrophage activity. Results: compared with the untreated uterine macrophages produced by low dose mifepristone treatment of uterine macrophages in IL-12p70, IL-23 and IL-p40 increased significantly, while the expression of IL-10 was significantly decreased; and compared with the 65nmol/L treatment by mifepristone uterine macrophages. The 200nmol/L of mifepristone treatment of uterine macrophages produced significantly More IL-12p70, IL-23 and IL-12p40, and the inhibition of IL-10 production function is more obvious. The low dose mifepristone treatment among the untreated macrophages and macrophage cell viability had no significant difference. Compared with mifepristone treated macrophages by 200nmol/L, high IL-12p70, progesterone to induce macrophages to produce 200nmol/L of mifepristone IL-23, IL-p40 the expression has no effect, but does not affect the inhibition of 200nmol/L of mifepristone on the expression of IL-10 in macrophages. Dexamethasone can inhibit 200nmol/L induced by mifepristone in macrophages produced IL-12p70, expression of IL-23 and IL-12p40, and can inhibit the reverse 200nmol/L of mifepristone on the expression of IL-10 in macrophages. Conclusion: Mifepristone promotes the transformation of uterine macrophages from M2 type to M1 type by glucocorticoid hormone receptor antagonism, which may enhance the immune Th1 The response of Th2 and inhibit the immune response, resulting in the failure of embryo implantation, which may be one of the mechanisms of contraception mifepristone. In the third part, through the activation of TAK1 promotes M1 macrophage activation Objective: to clear the molecular mechanism of mifepristone on uterine macrophage activation. Methods: high purity separation to the body outside the uterine macrophages by 200 nmol/L mifepristone, 200 nmol/L mifepristone 6umol/L and dexamethasone treatment. NF kappa beta, detected by Western blotting with p38 MAPK, ERK, changes of JNK and phosphorylation of TAK1 protein. Using NF kappa beta, p38MAPK and TAK1 specific inhibitor of JSH-23, SB203580 and 5Z-7-oxozeaenol to detect the effect of M1 type macrophage mediated activation of mifepristone. Results: after 200nmol/L treatment of mifepristone uterine macrophages I kappa beta alpha phosphorylation was significantly increased, 6umol/L dexamethasone can significantly inhibit the Effect of mifepristone. And inhibition of I mediated by Beta Kappa alpha phosphorylation, dexamethasone also inhibited the mifepristone mediated I Beta Kappa a degradation. In addition, dexamethasone inhibits cell core translocation of phosphorylated p65 mifepristone mediated by 200nmol/L. The treatment of mifepristone uterine macrophages p38 MAPK phosphorylation increased significantly, dexamethasone significantly inhibition of the.JSH-23 induced by mifepristone can significantly inhibit the IL-12p70 expression of IL-23 and IL-12p40, but JSH-23 did not increase the expression of.SB203580 IL-10 inhibited p38MAPK activation of mifepristone induced macrophage activation had no effect. The classic 200nmol/L mifepristone treatment of uterine macrophage TAK1 phosphorylation was significantly increased, dexamethasone can significantly inhibit the effect of.5Z-7-oxozeaenol can significantly inhibit NF kappa beta activation induced by mifepristone, and inhibit IL- The production of 12p70, IL-23 and IL-12p40 can significantly increase the expression level of IL-10 at the same time. Conclusion: mifepristone can turn the macrophages from M2 to M1 type by activating TAK1, which may lead to failure of embryo implantation, so as to achieve the effect of contraception.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R979.21

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