本文選題：子宮　切入點：巨噬細胞　出處：《浙江大學(xué)》2017年博士論文

【摘要】：藥物避孕是婦女避孕史上的里程碑。自上世紀60年代以來,甾體激素類口服避孕藥(Oral contraceptives,OCs),已在全球得到了廣泛的應(yīng)用。但是由于口服避孕藥含有雌激素,存在導(dǎo)致血栓性疾病、激素依賴性腫瘤及體重增加等副作用,限制了它的使用;而單純孕激素類避孕藥又由于存在不規(guī)則陰道流血及月經(jīng)周期控制欠佳等缺點,影響了它在臨床上的應(yīng)用。因此開發(fā)和研制新一代口服避孕藥,避免和減少上述的這些不良反應(yīng)迫在眉睫。米非司酮是一種具有強效的抗孕激素受體和抗糖皮質(zhì)激素受體特性的合成19-去甲睪酮衍生物,自其合成以來在臨床上得到廣泛應(yīng)用,包括婦產(chǎn)科學(xué)、腫瘤學(xué)、免疫學(xué)及內(nèi)分泌學(xué)等。目前,米非司酮也是一種在無保護性生活之后、高效的緊急避孕方法,并且已經(jīng)有研究認為其有潛力成為新型的、副作用極少的長期口服避孕藥;研究已經(jīng)表明米非司酮并不直接對胚胎產(chǎn)生不利影響,但可通過作用于內(nèi)膜來防止胚胎種植而又不影響排卵,從而實現(xiàn)"內(nèi)膜避孕"。但是,米非司酮避孕的確切作用機制仍有待進一步闡明。巨噬細胞是可塑性最強的免疫細胞,存在于所有組織中,并且可以根據(jù)它們所處微環(huán)境的變化而改變其功能。由于在人類的子宮內(nèi)膜中幾乎沒有樹突狀細胞,巨噬細胞就成為了最主要的抗原呈遞細胞。子宮巨噬細胞可能在胚胎成功植入所需的幾個過程中發(fā)揮關(guān)鍵作用,包括子宮內(nèi)膜的重塑、相鄰免疫細胞的免疫調(diào)節(jié)、滋養(yǎng)細胞侵襲和胎兒-母體免疫耐受的調(diào)節(jié)等。深入認識和探索米非司酮確切的避孕機理可能為新型避孕藥的開發(fā)和研制提供科學(xué)依據(jù)。第一部分子官巨噬細胞在基礎(chǔ)狀態(tài)下為M2型巨噬細胞目的:明確子宮巨噬細胞在基礎(chǔ)狀態(tài)下的活化表型。方法:從早孕期婦女外周血分離單核細胞,體外分別誘導(dǎo)分化成經(jīng)典活化(M1)型巨噬細胞及替代活化(M2)型巨噬細胞;同時從早孕期蛻膜組織分離、培養(yǎng)得到高純度的子宮巨噬細胞,在顯微鏡下觀察子宮巨噬細胞的形態(tài),采用流式細胞術(shù)檢測子宮巨噬細胞的CD14+表面標(biāo)記。采用ELISA檢測子宮巨噬細胞培養(yǎng)的上清液中的細胞因子IL-12p70、IL-23、IL-p40及IL-10的表達水平,將其分別與M1型巨噬細胞及M2型巨噬細胞分泌的相應(yīng)的細胞因子相比較。結(jié)果:在本研究中,我們采用免疫磁珠分選法成功分離到了高純度的子宮巨噬細胞(90%以上);與M1型巨噬細胞相比,子官巨噬細胞分泌的抗炎因子IL-10水平明顯更高,表達的促炎因子IL-12p70、IL-23和IL-p40水平明顯低下,而子宮巨噬細胞與M2巨噬細胞表達的細胞因子相似,即高IL-10水平和低IL-12p70,IL-23和IL-p40水平。結(jié)論:子宮巨噬細胞在基礎(chǔ)狀態(tài)下呈現(xiàn)M2表型,這可能有利于胚胎種植及妊娠的發(fā)展。第二部分米非司酮通過糖皮質(zhì)激素受體拮抗作用促進M1型巨噬細胞活化目的:明確米非司酮對子宮巨噬細胞活化的作用及途徑。方法:將體外分選到的高純度子宮巨噬細胞采用不同濃度的米非司酮、米非司酮+孕酮、米非司酮+地塞米松進行處理。采用ELISA檢測米非司酮、米非司酮+孕酮、米非司酮+地塞米松對子官巨噬細胞活化的影響。采用MTT檢測米非司酮對子宮巨噬細胞活力的影響。結(jié)果:與未處理的子宮巨噬細胞相比,經(jīng)低劑量米非司酮處理的子宮巨噬細胞產(chǎn)生的IL-12p70、IL-23和IL-p40明顯增加,而IL-10的表達水平卻明顯降低;并且與經(jīng)65nmol/L米非司酮處理的子宮巨噬細胞相比,經(jīng)200nmol/L米非司酮處理的子宮巨噬細胞明顯產(chǎn)生更多的IL-12p70、IL-23和IL-12p40,而抑制IL-10產(chǎn)生的作用更加明顯。經(jīng)低劑量米非司酮處理的巨噬細胞與未經(jīng)處理的巨噬細胞之間的細胞活力沒有顯著性差異。與經(jīng)200nmol/L米非司酮處理的巨噬細胞相比,孕酮對200nmol/L米非司酮誘導(dǎo)巨噬細胞產(chǎn)生的高IL-12p70、IL-23、IL-p40表達沒有影響,而且不影響200nmol/L米非司酮對巨噬細胞IL-10表達的抑制。地塞米松能明顯抑制200nmol/L米非司酮誘導(dǎo)巨噬細胞產(chǎn)生的IL-12p70、IL-23和IL-12p40的表達水平,同時能逆轉(zhuǎn)200nmol/L米非司酮對巨噬細胞IL-10表達的抑制。結(jié)論:米非司酮通過糖皮質(zhì)激素受體拮抗作用促進子宮巨噬細胞從M2型向M1型轉(zhuǎn)化,從而有可能增強Th1免疫應(yīng)答而抑制Th2免疫應(yīng)答,最終導(dǎo)致胚胎植入失敗,這可能是其避孕機理之一。第三部分米非司酮通過激活TAK1促進M1型巨噬細胞活化目的:明確米非司酮對子宮巨噬細胞活化的分子機制。方法:將體外分選到的高純度子宮巨噬細胞采用200 nmol/L米非司酮、200 nmol/L米非司酮和6umol/L地塞米松進行處理。采用蛋白免疫印跡法檢測NFκβ、p38 MAPK、ERK、JNK及TAK1蛋白磷酸化水平的變化。分別采用NFκβ、p38MAPK及TAK1特異性抑制劑JSH-23、SB203580及5Z-7-oxozeaenol檢測其對米非司酮介導(dǎo)的M1型巨噬細胞活化的影響。結(jié)果:經(jīng)200nmol/L米非司酮處理的子宮巨噬細胞Iκβα的磷酸化明顯增加,6umol/L地塞米松能明顯抑制這一作用。與抑制米非司酮介導(dǎo)Iκβα磷酸化一致,地塞米松也能抑制米非司酮介導(dǎo)的Iκβa降解。另外,地塞米松抑制了米非司酮介導(dǎo)的磷酸化p65的細胞內(nèi)核轉(zhuǎn)位。經(jīng)200nmol/L米非司酮處理的子宮巨噬細胞p38 MAPK的磷酸化明顯增加,地塞米松能明顯抑制這一作用。JSH-23能明顯抑制米非司酮誘導(dǎo)的IL-12p70、IL-23和IL-12p40的表達水平,但是JSH-23并不能增加IL-10的表達水平。SB203580抑制p38MAPK激活后對米非司酮誘導(dǎo)的經(jīng)典巨噬細胞活化沒有影響。經(jīng)200nmol/L米非司酮處理的子宮巨噬細胞TAK1的磷酸化明顯增加,地塞米松能明顯抑制這一作用。5Z-7-oxozeaenol能明顯抑制米非司酮誘導(dǎo)的NFκβ活化,并且明顯抑制IL-12p70、IL-23和IL-12p40的產(chǎn)生,同時能明顯增加IL-10的表達水平。結(jié)論:米非司酮通過激活TAK1將子宮巨噬細胞從M2型轉(zhuǎn)向M1型,可能導(dǎo)致胚胎種植失敗,從而達到避孕的效果。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R979.21

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