【作者】 陈悦洲；

【导师】 黄丽丽；

【作者基本信息】 浙江大学 ， 妇产科学， 2012， 博士

【摘要】 米非司酮是一种人工合成的19-去甲基睾酮衍生物,具有抗孕激素和糖皮质激素作用。从首次合成米非司酮以来,米非司酮已被广泛用作抗早孕治疗,并成功用于紧急避孕。多年来的基础和临床研究表明,米非司酮在妇产科学、内分泌学、肿瘤学及免疫学等多个领域表现出一定的应用潜力。近年的研究表明,米非司酮对女性的排卵、激素水平以及月经出血模式没有明显影响,但是能够延缓子宫内膜的成熟,从而达到内膜避孕的作用。然而,米非司酮内膜避孕的作用机制还不十分清楚。第一部分人子宫内膜间质细胞对uNK细胞体外存活及功能的影响目的：明确人子宫内膜间质细胞对uNK细胞体外存活、增殖及毒性功能的影响。方法：分离不同时期女性子宫内膜和蜕膜间质细胞,体外培养制备间质细胞条件培养液。从早孕蜕膜组织中分离、分选得到高纯度的人uNK细胞,采用流式细胞方法检测细胞的表型。在光镜下观测了不同时期间质细胞条件培养液对uNK细胞形态学的影响；采用基于乳酸脱氢酶活性为基础的MTS试剂盒检测不同时期间质细胞条件培养液对uNK细胞增殖的影响,并采用流式细胞方法检测uNK细胞对靶细胞K562的细胞杀伤作用。结果：在本实验中,采用细胞免疫磁珠分选方法可以得到高纯度的人uNK细胞(94.52±2.44%)。外源性IL-2能够明显维持uNK细胞的体外存活,并对靶细胞的细胞毒性有促进作用。单独的不同时期间质细胞条件培养液均不能维持uNK细胞的体外存活,但分泌期和早孕期间质细胞条件培养基能够明显增加uNK细胞体外增殖(1.12±0.03和1.22±0.06,p<0.05)。与对照组相比(63.39±±5.18%),不同时期间质细胞条件培养液均能明显降低uNK细胞毒性功能(46.98±±3.58%,45.28±±4.05%,43.72±±1.67%,p<0.05),而这种抑制作用在不同时期间质细胞条件培养液间并没有显著差异(p>0.05)。结论：人子宫间质细胞条件培养基不能维持uNK细胞的体外存活,但能够通过细胞增殖和毒性的影响来调节uNK细胞功能。第二部分米非司酮对人子宫NK细胞生物活性的影响目的：研究米非司酮对人uNK细胞体外增殖、凋亡、IFN-γ分泌、细胞毒性功能及穿孔素和颗粒酶-B蛋白表达水平的影响。方法：体外分选得到的高纯度人uNK细胞,采用不同浓度米非司酮进行体外处理。采用MTS试剂盒检测细胞增殖情况；采用流式细胞方法检测细胞凋亡、毒性功能及毒性颗粒的表达情况；ELISA试剂盒检测细胞IFN-γ分泌水平。结果：不同浓度米非司酮对人uNK细胞的体外增殖、凋亡及IFN-γ的分泌功能均没有明显影响。经65nmol/L和200nmol/L的米非司酮处理后,uNK细胞的毒性分别为68.92±5.96%和69.62±4.22%,虽然高于对照组(62.24±4.39%),但没有显著性差异(p>0.05)；1000nmol/L的米非司酮能够明显增加其细胞毒性(73.16±4.27%,p<0.05)。1000nmol/L米非司酮不会影响人uNK细胞颗粒酶-B的蛋白表达(50.50±0.02%和45.97±0.03%,p>0.05),但能够增加穿孔素的蛋白表达水平(49.13±0.03%和36.23±0.05%,p<0.05)。结论：米非司酮对人uNK细胞的体外增殖、凋亡及IFN-γ的分泌功能均没有影响,但可能通过对穿孔素表达水平的上调作用来增加uNK细胞的毒性功能。第三部分米非司酮在人子宫NK细胞毒性调节中的作用及机制目的：研究米非司酮的糖皮质激素拮抗效应在人uNK细胞毒性功能和穿孔素蛋白表达调节中的作用及可能的信号途径。方法：体外分离得到的高纯度人uNK细胞,分别采用不同浓度的孕激素、皮质醇以及米非司酮进行体外处理。采用流式细胞方法检测了uNK细胞毒性功能和穿孔素蛋白的表达情况；采用免疫蛋白印迹检测了ERK1/2、p38和JNK蛋白磷酸化水平的变化；采用ERK1/2蛋白活化的特异性抑制剂PD98059检测了ERK1/2活性在米非司酮诱导的uNK细胞毒性功能和穿孔素表达调节中的作用。结果：不同浓度的孕激素对uNK细胞毒性没有明显影响；而1×10-6M和1×10-5M的皮质醇能够明显降低uNK细胞的细胞毒性(55.07±4.04%、56.20±3.11%和62.30±2.69%,p<0.05)。1×10-6M的米非司酮能够促进uNK细胞毒性功能(62.30±2.69%和73.16±4.26%,p<0.05)和穿孔素蛋白的表达(36.23±4.84%和49.13±2.92%,p<0.05)；1×10-6M皮质醇能够抑制米非司酮对uNK细胞毒性功能(73.16±4.26%3和66.92±2.87%,p<0.05)和穿孔素蛋白表达的诱导(49.13±2.92%和33.14±3.45%,p<0.05)。1×10-6M的米非司酮能够增加ERK1/2蛋白的磷酸化水平,但对p38和JNK蛋白磷酸化水平没有明显影响；皮质醇对米非司酮诱导的ERK1/2蛋白的磷酸化水平具有抑制作用(0.87±0.09和0.48±0.07,p<0.05)。此外,PD98059能够明显降低米非司酮对uNK细胞毒性和穿孔素蛋白表达的诱导作用。结论：米非司酮通过糖皮质激素拮抗效应来促进人uNK细胞毒性功能和穿孔素的表达,并诱导ERK1/2蛋白的磷酸化。ERK1/2信号途径可能参与了米非司酮对uNK细胞毒性功能和穿孔素表达的调节。更多还原

【Abstract】 Mifepristone (RU486) is a synthetic19-norsteroid, which is a potent antagonist of progesterone and glucocorticoid. When it was first found, mifepristone has been used as an abortion drug. Accumulating evidences from the basic and clinical researches demonstrated a variety of potential applications for mifepristone in the areas of gynecology, endocrinology, oncology, and immunology. Recently, several studies have shown that low-dose mifepristone retards endometrial development to achieve the purpose of contraception, so-called endometrial contraception. However, the exact mechanisms responsible for contraception by mifepristone are not fully understood.Part I Effects of human uterine stromal cells on uNK-cell survival and function in vitro Objective:To investigate the effects of human uterine stromal cells on uNK-cell survival, proliferation and cytotoxicity in vitro.Methods:Human endometrial stromal cells were separated in vitro. The conditioned medium was derived from the endometrial stromal cells in proliferative phase, secretory phase, and early pregnancy. Uterine natural killer (uNK) cells were separated and purified from human decidual samples of early pregnancy and analysed by flow cytometer. The effects of stromal cell-derived conditioned medium on the morphology of uNK cells were detected by light microscope. uNK-cell proliferation and cytotoxicity to target cells K562were examined by mitochondrial lactate dehydrogenase-based MTS staining and flow cytometry.Results:In current study, the high purity of uNK cells could be purified by using the human NK-cell immune magnetic beads isolation system (94.52±2.44%). Recombination IL-2could maintain uNK-cell survival and promote uNK-cell cytotoxicity. Conditioned medium from stromal cells at different physiological stages could not maintain uNK-cell survival in vitro; conditioned medium from stromal cells in both the secretory phase and early pregnancy could promote uNK-cell proliferation (1.12±0.03and1.22±0.06). In contrast with the control group (63.39±5.18%), conditioned medium in all groups were able to inhibit uNK-cell cytotoxicity (46.98±3.58%,45.28±4.05%,43.72±1.67%). However, there was no significant difference among the proliferation, secretory, and decidua groups.Conclusion:Human endometrial stromal cells could not maintain the uNK-cell survival, but may be involved in the regulation of uNK-cell functions through influencing proliferation and cytotoxicity. Part Ⅱ Effects of mifepristone on the bioactivity of human uNK cellsObjective:To investigate the influences of different doses of mifepristone on the proliferation, apoptosis, IFN-γ secretion, and the expression of perforin and granzyme-B in human uNK cells.Methods:Human uNK cells were purified from decidual tissue and treated with different concentrations of mifepristone in vitro. The cell proliferation was examined by using MTS staining; flow cytometer was used to evaluate the apoptosis, cytotoxicity and expression of perforin and granzyme-B; ELISA assay was used to detect the secretion level of IFN-γ.Results:We found that mifepristone (0,65,200and1000nmol/L) dose not directly affect the proliferation, apoptosis, and IFN-γ secretion of human uNK cells.65nmol/L and200nmol/L mifepristone had no significant influence on uNK cell-mediated cytotoxicity (68.92±5.96%,69.62±4.22%and62.24±4.39%, p>0.05), which could be significantly augmented by1000nmol/L mifepristone (62.24±4.39%and73.16±4.27%, p<0.05). In addition,1000nmol/L mifepristone significantly promoted the expression level of perforin (49.13±0.03%and36.23±0.05%, p<0.05) but not granzyme-B (50.50±0.02%and45.97±0.03%, p>0.05).Conclusion:Our current study indicated that mifepristone dose not directly affect the proliferation, apoptosis, and IFN-y secretion of uNK cells, but augments uNK-cell cytotoxicity and this effect is probably due to the increased expression of perforin by mifepristone. Part Ⅲ The roles and mechanisms of mifepristone in the regulation of human uNK-cell cytotoxicityObjective:To explored whether the increased cytotoxicity and perforin expression in uNK cells by mifepristone is due to either anti-progesterone or anti-glucocorticoid activity, and also investigated relevant changes in the mitogen-activated protein kinase (MAPK) pathway and evaluated the roles of ERKl/2activity in mifepristone-induced changes of human uNK-cell cytotoxicity and perforin expression.Methods:Human uNK cells were purified from decidual tissue and treated with different concentration of progesterone, cortisol and mifepristone in vitro. Flow cytometer was used to examine the cytotoxicity and perforin expression of uNK cells; Western blotting was used to detect the phosphorylation level of ERK1/2, p38and JNK. A specific ERK1/2inhibitor of the MAPK pathway, PD98059, was used to evaluate the roles of ERK1/2activity in the mifepristone-induced changes of human uNK-cell cytotoxicity and perforin expression.Results:Progesterone had no effects on the cytotoxicity of human uNK cells and cortisol (1×10-6M and1×10-5M) significantly decreased in the cytotoxicity of uNK cells (55.07±4.04%,56.20±3.11%and62.30±2.69%, p<0.05). Mifepristone (1×10-6M) increased the uNK cell-mediated cytotoxicity (62.30±2.69%and73.16±4.26%, p<0.05) and perforin expression (36.23±4.84%and49.13±2.92%, p<0.05) and these effects could be reversed by cortisol (1×10-6M)(73.16±4.26%3and66.92±2.87%, p<0.05;49.13±2.92%and33.14±3.45%, p<0.05). Mifepristone increased the phosphorylation levels of ERK1/2but not p38and JNK in uNK cells; cortisol significantly suppressed the mifepristone-induced phosphorylation of ERK (0.87±0.09and0.48±0.07, p<0.05). After pretreatment with PD98059, mifepristone had no effect on uNK cell-mediated cytotoxicity and perforin expression, with or without cortisol.Conclusion:Mifepristone augments the uNK-cell cytotoxicity and increases the perforin expression through the antagonism of glucocorticoid, and induces the phosphorylation of ERK1/2in human uNK cells. ERK1/2pathway may be involved in the immune regulation of human uNK cells by mifepristone.更多还原