【作者】 喻静；

【导师】 李大金； 王凌；

【作者基本信息】 复旦大学 ， 妇产科学， 2008， 博士

【摘要】 子宫内膜异位症发病机制复杂,其中Sampson的经血逆流学说已获得普遍认可。正常情况下约有50%～90%的育龄妇女发生经血逆流,但只有6.2%～8.2%罹患EMs。因此,EMs患者可能存在浆膜免疫失调,导致难以清除逆流的内膜碎屑。大量研究证据表明,EMs患者的外周及腹腔细胞免疫功能改变,从而降低了对异位子宫内膜的免疫监视、识别和清除,可能促进子宫内膜的异位种植。在子宫内膜异位症的发生和发展过程中,大量免疫细胞被募集到腹膜腔内,特别是单核巨噬细胞、T细胞亚群及其功能改变。单核巨噬细胞在免疫应答中起关键作用,巨噬细胞非但不能有效清除逆流子宫内膜;反而通过分泌诸如促炎因子、生长因子及血管形成因子,促进其在腹腔内粘附和生长。因此,单核巨噬细胞在子宫内膜异位症发病中具有重要促进作用。炎性细胞在趋化因子的浓度梯度介导下,从血循环进入组织。异位灶炎性细胞可能由种植部位的细胞合成释放的特定趋化因子介导。从分析单核细胞的趋化因子受体表达谱入手,筛选出EMs关联的趋化因子受体,从而有助于解析趋化因子通过募集单核巨噬细胞,介导EMs发病的分子机制。EMs的发病受多种因素影响,一方面,EMs是一种雌激素依赖性疾病;另一方面,环境污染物参与EMs的发病。环境因素可能是人类EMs发病率升高的重要因素。含碳和氯燃料所产生的二噁报（TCDD）是全球普遍存在的最严重的大气污染物,可在大气中长期稳定存在。许多研究证明TCDD与EMs的发病具有相关性。TCDD的亲脂特性决定其不能从尿中排泄而在体内脂肪中长期堆积,作用机制与激素类似,通过与细胞内特异受体结合形成复合物,作为转录因子调控多种蛋白质分子生成。TCDD具有抗雌激素和雌激素样双重作用;随着年龄、月经周期、组织和其它因素变化所致的雌激素水平,决定着TCDD发挥雌激素效应,或抗雌激素效应。本研究假设是:环境污染物TCDD与体内雌激素协同作用,导致腹腔内分泌及免疫微环境改变。因此,我们通过体外构建异位灶模型,探索17β-E₂和TCDD通过调控趋化因子的分泌,募集单核巨噬细胞的确切机制,及其在EMs发生与发展中的作用及意义。1、单核细胞系U937趋化因子受体表达特征及17-β雌二醇和二噁英（TCDD）对其调节作用应用RT-PCR方法分析单核细胞系U937细胞18种趋化因子受体的表达谱。并观察17β-E₂和TCDD对相应趋化因子受体表达的影响。结果发现,单核细胞系U937细胞表达所有18种趋化因子受体。其中,高表达CCR5,CCR9,XCR1及CX3CR1;中等强度表达CCR4,CCR7及CXCR4;其余趋化因子均有不同程度的弱表达。使用不同的浓度17β-雌二醇和/或二噁英处理U937细胞,随着17β-雌二醇及TCDD浓度的增加,U937细胞的CCR1的mRNA转录逐渐升高,17β-雌二醇联合TCDD进一步促进CCR1的转录。与CCR1相反,17β-雌二醇及TCDD下调CCR10的mRNA的表达。TCDD明显降低CCR6mRNA的表达;但17β-雌二醇对此无明显影响。高浓度的17β-雌二醇（10-7M）下调CXCR4mRNA的表达;而TCDD及两者联合对CXCR4mRNA的表达则无明显影响。17β-雌二醇或/和TCDD对趋化因子受体CCR2,CCR3,CCR4,CCR5,CCR7,CCR8,CCR9,CXCR1,CXCR2,CXCR3,CXCR5,CXCR6和CX3CR1mRNA的表达无明显影响。经用Western法研究证实,17β-雌二醇和TCDD对CCR1,CCR5蛋白表达的影响与mRNA结果一致。综上所述,雌激素与TCDD通过调控单核细胞趋化因子受体的表达,可能参与子宮内膜异位症的发病。2.17β-雌二醇及二噁英对异位灶关联细胞表达CCR1、CCR5/RANTES、MIP-1α的调控作用在建立了不同的异位灶关联细胞直接接触或间接接触共培养体系后,观察17β-雌二醇及二噁英对共培养体系CCR1、CCR5/RANTES、MIP-1α表达的调控作用。我们使用ELISA方法检测RANTES和MIP-1α的分泌;应用Western分析U937细胞CCR1,CCR5的表达。ESC-HPMC共培养促进RANTES分泌。HPMC-U937共培养体系的RANTES分泌水平明显高于ESC-U937和ESC-HPMC共培养体系。三者共培养进一步促进RANTES的分泌,其中以三者直接接触共培养（U-E-H）RANTES分泌水平最高。在三种间接共培养体系中,E/H-U体系的RANTES分泌量明显高于U/H-E和H/U-E体系。同时,三者共培养体系与相应两者共培养体系相比,U/H-E,H/E-U,E/H-E的RANTES分泌水平均明显高于H-E,E-U,H-E。17β-雌二醇联合二噁英明显促进E/H-U,H/E-U共培养体系的RANTES分泌。HPMC-U937两者共培养体系的MIP-1α分泌水平明显高于ESC-U937和ESC-HPMC共培养体系。三者共培养进一步促进MIP-1α的分泌,其中以三者直接接触共培养（U-E-H）分泌水平最高。在三种间接共培养体系中,E/H-U体系的MIP-1α分泌水平明显高于U/H-E和H/U-E体系。同时,三者共培养体系与相应两者共培养体系相比,U/H-E,H/E-U,E/H-E的MIP-1α分泌水平均明显高于H-E,E-U,H-E。17β-雌二醇联合二噁英明显促进了E/H-U共培养体系的MIP-1α的分泌。以上结果表明,异位灶关联细胞共培养明显促进了趋化因子RANTES,MIP-1α的分泌。在所有共培养体系中,17β-雌二醇联合TCDD均促进U937细胞CCR1的表达。17β-雌二醇联合TCDD仅上调E/H-U共培养体系CCR5的表达。本研究提示,17β-E₂和TCDD通过对趋化因子RANTES和MIP-1α以及相应受体CCR1,CCR5的表达,可能引导单核巨噬细胞向腹腔内募集,促进内异症腹腔内的炎症反应,加重病情发展。3.17β-雌二醇及二噁英通过上调CC类趋化因子介导单核细胞的趋化及ESC的侵袭通过趋化试验我们研究发现,ESC-HPMC共培养体系促进了单核细胞的趋化;RANTES和MIP-1α中和抗体部分抑制这种趋化作用。17β-雌二醇联合TCDD也促进U937细胞对ESC的趋化;RANTES、MIP-1α、CCR1和CCR5的中和抗体能部分抑制了这种趋化作用。因此,17β-雌二醇与TCDD协同作用,通过升调RANTES,MIP-1α/CCR1,CCR5的表达,促进U937细胞的趋化。进一步研究证实,ESC/HPMC-U937共培养增加了ESC的侵袭及MMP2,9的活性;RANTES,MIP-1α/CCR1,CCR5中和抗体可以部分抑制这种效应。17β-雌二醇联合TCDD促进ESC细胞的侵袭,及MMP 2,9的蛋白表达及活性;RANTES,MIP-1α/CCR1,CCR5中和抗体也可以部分抑制这种效应。另外,hrRANTES和hrMIP-1α可以促进ESC的侵袭性。因此,本部分实验表明趋化因子RANTES和MIP-1α介导单核细胞的趋化并促进ESC的侵袭。17β-雌二醇与合TCDD协同作用,亦通过升调RANTES,MIP-1α/CCR1,CCR5促进单核细胞的趋化及ESC的侵袭。综上所述,单核细胞表达所有18种趋化因子受体。17β-雌二醇与TCDD促进单核巨噬细胞趋化因子受体CCR1的表达。异位灶关联细胞共培养体系促进了趋化因子RANTES、MIP-1α的分泌。17β-雌二醇与TCDD协同作用促进了E/H-U共培养体系RANTES、MIP-1α的分泌,且促进这种共培养体系的U937细胞的CCR5及5种共培养体系的CCR1的表达。17β-雌二醇与TCDD通过升调RANTES,MIP-1α/CCR1,CCR5趋化因子配受体对,促进了单核细胞的趋化及ESC的侵袭力。更多还原

【Abstract】 The pathogenesis of endometriosis remains to be elucidated.According to Sampson’s transplantation theory,the pelvic endometriosis is initiated by the retrograde menstruation when menstrual fragments flow out of the fimbriated end of the fallopian tubes,and become established on the ovarian surface or other sites in the peritoneal cavity.Since the retrograde menstruation is a physiological process occurring in fertile women,other factors must be involved in the disease.Alteration/ dysfunction of the immune system may lead to the development of endometriosisCurrent evidence suggests the peritoneal immuno-inflammation might be associated with the origin of endometriosis.During the development of endometriosis, the immune cells are recruited into the peritoneal cavity,and among them macrophages are the dominant cell type that are involved in phagocytosis and inflammation,especially in cleaning the retrograded endometrial debris.The peritoneal macrophages isolated from patients with endometriosis have been found to have phenotypic and functional alterations leading to poor phagocytotic capacity, which is highly associated with severity of endometriosis.The peritoneal macrophages may improve pathogenesis of endometriosis by secreting many sorts of cytokines,and thereby play a very important role in endometriosis.Chemokine is required in the process of recruiting circulating leukocytes into pelvic sites. Chemokine activity is mediated through cell surface chemokine receptors,and the functions of chemokine depend on binding to the corresponding receptors.To explore the role of chemokine in the pathogenesis of endometriosis,it is facilitated to investigate chemokine as well as its receptor because of the complexity of the interaction between the chemokines and their receptors.To pick up chemokine receptors as well as their ligands may provide some useful insights into the molecular mechanisms of the pathogenesis of endometriosis.So we evaluated 18 chemokine receptors of U937 cells,which would help us to explore pathogenesis of endometriosis.Endometriosis is an estrogen-depending disease.Recently,environmental contaminants have been also suggested to play a role in the pathogenesis of endometriosis.Research on nonhuman primates has shown that exposure to the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin（TCDD） is associated with an increased prevalence and severity of endometriosis.Investigating the regulatory mechanism of dioxin and estrogen in chemokines and their receptors expression will throw a light in elucidating the role of the chemokines in the etiology of endometriosis.1.The expression characteristics of chemokine receptors in U937 cells and the modulating effect of 17β-estradiol and TCDD on the expressed chemokine receptorsThe transcriptions of 18 chemokine receptors in U937 cells were first analyzed by semi-quantified RT-PCR.Among 18 chemokine receptors,CCR5,CCR9,XCR1 and CX3CR1 were highly expressed in U937 cells,while CCR4,CCR7 and CXCR4 were moderately expressed.Other chemokine receptors were lowly expressed.Both 17β-estradiol and TCDD increased the transcription and translation of CCR1.The combination of 17β-estradiol with TCDD further promoted the transcription and translation of CCR1.The 17β-estradiol or/and TCDD had no significant effect on transcription of CCR2,CCR3,CCR4,CCR5,CCR7,CCR8,CCR9,CXCR1, CXCR2,CXCR3,CXCR5,CXCR6 and CX3CR1 mRNA.The 17β-estradiol had no significant effect on transcription of CCR6,while TCDD significantly decreased expression of CCR6 mRNA.The 17β-estradiol or/and TCDD significantly decreased transcription of CCR10 mRNA.The 17β-estradiol of 10^-7M decreased expression of CXCR4 mRNA,but TCDD or the combination of 17β-estradiol with TCDD had no effect on expression of CXCR4 mRNA.The results above suggest that the effect of 17β-estradiol on chemokine receptors expression in U937 cells is one of mechanisms involved in pathogenesis of endometriosis.TCDD not only influences development of the disease as a pro-inflammatory factor but also produces a marked effect through the signal transduction of estrogen.The interaction of 17β-estradiol with TCDD were very complicated.Women with endometriosis were influenced by the internal and external environment,and their immune system were changed,which may be one important mechanism of onset of this disease.Chemokine receptor CCR1 of U937 cells may play an important role in pathogenesis of endometriosis.2.The modulating effect of 17β-estradioi and TCDD on expression of CCR1, CCR5/RANTES,MIP-1αby the endometriosis-associated cellsWe established different contact and non-contact co-culture model of the corresponding ectopic cells such as ESC,HPMC and U937 cells,and explored the effect of 17β-estradiol or/and TCDD on expression of CCR1,CCR5/RANTES, MIP-1αby using ELISA and Western blot.The co-culture of ESC and HPMC promoted secretion of RANTES.The RANTES secretion in co-culture of HPMC-U937 were significantly higher than the co-culture of ESC-U937 or ESC-HPMC,and the co-culture of three cells further promoted secretion of RANTES. The contact co-culture of U-E-H secreted highest RANTES.Among the three non-contact co-culture of three cells,secretion of RANTES in co-culture of E/H-U was significantly higher than the other two non-contact co-culture.Moreover,we found that the non-contact co-culture the three cells of U/H-E,H/E-U,E/H-E secreted more RANTES than the co-culture of two cells,H-E,E-U,H-E,respectively.The combination of 17β-estradiol with TCDD significantly increased secretion of RANTES of co-culture of E/H-U and H/E-U.The MIP-1αsecretion in the co-culture of HPMC-U937 was significantly higher than the co-culture of ESC-U937 or ESC-HPMC.The co-culture of three cells further promoted secretion of MIP-1α.The contact co-culture of U-E-H secreted highest MIP-1α.Among the three non-contact co-culture of three cells,secretion of MIP-1αof co-culture of E/H-U was significantly higher than the other two non-contact co-culture.It was found that the non-contact co-culture the three cells of U/H-E,H/E-U,E/H-E secreted more MIP-1αthan co-culture of two cells of H-E,E-U,H-E respectively.The combination of 17β-estradiol with TCDD increased the secretion of RANTES in the co-culture of E/H-U.Among all the co-culture units,the combination of 17β-estradiol with TCDD increased the expression of CCR1 in U937 cells.While the combination of 17β-estradiol with TCDD only increased the expression of CCR5 in U937 cells of E/H-U co-culture unit.The results above suggest that the combination of 17β-estradiol with TCDD promotes recruitment of mono-macrophages in peritoneal cavity by up-regulating expression of CCR1,CCR5/RANTES,MIP-1β,leading to the development of endometriosis. 3.Combination of 17β-estradiol with TCDD promoted recruitment of monocyte and invasion of ESC via CCR1,CCR5/RANTES,MIP-1αinteractionThe co-culture of ESC-HPMC promoted chemotaxis of U937 cells,which could be partly inhibited by anti-RANTES or anti-MIP-1αneutralizing antibody.The combination of 17β-estradiol with TCDD promoted the chemotaxis of U937 cells to ESC,which could be partly inhibited by anti-RANTES,anti-MIP-1α,anti-CCR1 or anti-CCR5 neutralizing antibody,which suggests the combination of 17β-estradiol with TCDD promotes chemotaxis of U937 cells via RANTES,MIP-1α/CCR1,CCR5 interaction.Invasion assay showed that the co-culture unit ESC/HPMC-U937 or the combination of 17β-estradiol with TCDD promoted ESC invasiveness and increased activity and protein expression of MMP 2,9 which could be partly inhibited by anti-RANTES,anti-MIP-1α,anti-CCR1 or anti-CCR5 neutralizing antibody. Meanwhile,both hrRANTES and hrMIP-1αcould promote invasiveness of ESC.The results above suggest that the combination of 17β-estradiol with TCDD promotes recruitment of monocyte and invasion of ESC via CCR1,CCR5/RANTES,MIP-1αinteraction.Collectively,it has been demonstrated in the present study that all the 18 chemokine receptors were expressed in U937 cells.The combination of 17β-estradiol with TCDD promotes expression of CCR1.The different co-culture units increase secretion of RANTES and MIP-1α,and the combination of 17β-estradiol with TCDD significantly promotes secretion of RANTES and MIP-1αof the co-culture unit of ESC/HPMC-U937.The combination of 17β-estradiol with TCDD promotes recruitment of monocyte and invasion of ESC via CCR1,CCR5/RANTES,MIP-1αinteraction.Our research suggests a possible mechanistic link between chemokine/chemokine receptor and endometriosis pathogenesis.更多还原