【作者】 张辉；

【导师】 温泽清；

【作者基本信息】 山东大学 ， 妇产科学， 2010， 博士

【摘要】 子宫内膜异位症是生育期女性常见疾病,以具有活性的子宫内膜组织出现在子宫内膜以外部位为特点,是一种雌激素依赖性疾病。以持续加重的盆腔粘连、疼痛、不孕为主要临床表现。目前关于异位子宫内膜来源的学说有多种,包括子宫内膜种植学说、淋巴及静脉播散学说、体腔上皮化生学说、诱导学说、遗传学说、免疫调节学说及血管生成等,但是其发病机制目前尚未完全明了。本研究通过临床标本及细胞培养,在组织及细胞水平研究了PI3K/PTEN/AKT/Survivin信号通路以及IκB/NFκB信号通路在子宫内膜异位症的存在及意义。研究目的①研究子宫内膜异位症腺上皮细胞中PI3K/PTEN/AKT信号通路、IκB/NFκB信号通路以及MAPKs/ERK1/2信号通路的活性状态；②研究雌激素对正常及子宫内膜异位症腺上皮细胞生长增殖的作用,以及PI3K/PTEN/AKT信号通路、IκB/NFκB信号通路、MAPKs/ERKl/2信号通路在该作用中的意义；③研究正常内膜及子宫内膜异位症在位及异位内膜中间质细胞对腺上皮细胞中存活／凋亡信号：PI3K/AKT/Survivin信号传导通路以及细胞生长增殖的调节作用；④研究正常及子宫内膜异位症内膜腺上皮细胞对正常子宫内膜间质细胞中基质金属蛋白酶(matrix metalloproteinases, MMPs)表达及活性的诱导作用,以及IκB/NFκB信号通路在该作用中的意义；研究方法①子宫内膜异位症在位及异位内膜、正常子宫内膜无菌新鲜组织标本取自手术过程中,被分为两部分,一部分甲醛固定后用于免疫组织化学分析；另一部分立即用于原代细胞的分离培养；②应用免疫组织化学方法检测正常及子宫内膜异位症在位内膜组织标本中PI3K/PTEN/AKT信号通路,IκB/NFκB信号通路状态；③原代细胞分离培养：手术中获取新鲜正常及子宫内膜异位症内膜组织,通过胶原酶消化法分离培养正常子宫内膜及子宫内膜异位症在位、异位内膜间质细胞和腺上皮细胞,并应用免疫细胞化学方法检测细胞中波形蛋白或角蛋白表达与否鉴定细胞来源；④通过应用Western Blot方法检测AKT和ERK1／2磷酸化水平来确定正常及子宫内膜异位症内膜腺上皮细胞中PI3K/PTEN/AKT信号通路和MAPKs/ERK1/2信号通路活性状态；应用凝胶电泳迁移率实验(EMSA)方法检测NFκBDNA-结合能力,从而确定正常及子宫内膜异位症内膜腺上皮细胞中IκB/NFκB信号通路的活性状态；⑤建立多种间质-腺上皮细胞共培养体系：通过将1：1比例的间质细胞和腺上皮细胞直接混合后接种于细胞培养板建立直接共培养体系；通过条件培养基、细胞裂解物以及Millipore transwell系统等方法建立间接共培养体系；通过直接及间接共培养两种方式,建立正常内膜间质-腺上皮细胞共培养体系,子宫内膜异位症在位内膜间质-腺上皮细胞共培养体系,子宫内膜异位症异位内膜间质-腺上皮细胞共培养体系,以及正常内膜间质-子宫内膜异位症在位或异位腺上皮细胞共培养体系,子宫内膜异位症异位间质-正常腺上皮细胞共培养体系；⑥应用免疫组织化学方法检测正常及子宫内膜异位症在位内膜中Survivn蛋白表达及组织定位；应用免疫细胞化学方法以及Western Blot方法检测原代分离培养的正常及子宫内膜异位症在位、异位腺上皮细胞中Survivin的蛋白表达及亚细胞定位；⑦应用Western Blot方法检测细胞中MMP2/MMP9及其组织型抑制物(tissue inhibitor of metalloproteinase, TIMP1/2)的表达及分泌；应用明胶酶谱分析检测细胞培养上清液中MMP2/MMP9的酶活性；⑧应用MTT法检测各种细胞在不同处理条件下的生长增殖情况。研究结果①显著性的AKT磷酸化水平增高及其通路抑制性调节分子PTEN表达水平降低存在于子宫内膜异位症在位内膜；②显著性的AKT磷酸化水平增高及其通路抑制性调节分子PTEN表达水平降低、ERK磷酸化水平增强和NFκBDNA-结合能力增强存在于原代分离培养的子宫内膜异位症在位及异位内膜腺上皮细胞；而MMP2/MMP9的表达和分泌明显增强也存在于原代分离培养的子宫内膜异位症在位及异位内膜腺上皮细胞；而正常及子宫内膜异位症间质细胞则仅表现杂较弱水平的MMP2/MMP9表达和分泌;③17β雌二醇可以明显促进正常及子宫内膜异位症在位、异位内膜腺上皮细胞中AKT和ERK1／2磷酸化以及NFκB的DNA-结合能力,同时可以降低细胞中PTEN蛋白及mRNA表达水平,促进细胞生长增殖；特异性PI3K/PTEN/AKT信号通路抑制剂LY294002、MAPKs/ERK1/2信号通路抑制剂U0126以及IκB/NFκB信号通路抑制剂PDTC可以显著性抑制正常及子宫内膜异位症在位、异位内膜腺上皮细胞中AKT和ERK1／2磷酸化,以及NFκB的DNA-结合能力,同时显著性地抑制了细胞的生长增殖；IκB／NFκB信号通路抑制剂PDTC同时也明显抑制了17β雌二醇对PTEN蛋白及mRNA表达的降低作用,而LY294002和U0126没有该作用；PI3K/PTEN/AKT信号通路抑制剂LY294002可以明显抑制正常及子宫内膜异位症在位、异位内膜腺上皮细胞中本底的及雌激素-诱导的NFκB的DNA-结合能力,而相反的,IκB/NFκB信号通路抑制剂PDTC也可以明显抑制细胞中本底的及雌激素-诱导的AKT磷酸化水平；而MAPKs/ERKl/2信号通路抑制剂U0126仅表现出对雌激素诱导的AKT磷酸化及NFκB的DNA-结合能力有较弱的抑制作用；同样,PI3K/PTEN/AKT信号通路抑制剂LY294002以及IκB/NFκB信号通路抑制剂PDTC也仅对雌激素诱导的ERK1／2磷酸化有抑制作用；④本研究发现,凋亡抑制蛋白Survivin表达存在于正常及子宫内膜异位症在位、异位内膜腺上皮细胞,且子宫内膜异位症在位及异位内膜腺上皮细胞常表现出较高水平的Survivin蛋白表达；PI3K/PTEN/AKT信号通路抑制剂LY294002可以明显抑制细胞中Survivin蛋白表达水平；在共培养体系中,产生于正常及子宫内膜异位症在位内膜间质细胞的条件培养基可以分别抑制相应腺上皮细胞中AKT磷酸化水平和Survivin蛋白表达,以及相应腺上皮细胞的生长增殖,而孕激素可以加强该抑制作用；产生于子宫内膜异位症异位内膜间质细胞的条件培养基不仅不可以抑制相应腺上皮细胞中AKT磷酸化水平和Survivin蛋白表达以及相应腺上皮细胞的生长增殖,也不能抑制正常腺上皮细胞中AKT磷酸化水平和Survivin蛋白表达以及细胞生长增殖；同时,本研究证明,在体外,分离培养的腺上皮细胞生长增殖能力强弱为：子宫内膜异位症在位内膜>异位内膜>正常内膜；⑤在共培养体系中,本研究发现来自于子宫内膜异位症在位、异位内膜腺上皮细胞的条件培养基及细胞裂解物可以在正常子宫内膜间质细胞诱导较高水平的MMP2/MMP9表达和分泌；使用IκB/NFκB信号通路抑制剂PDTC阻断子宫内膜异位症腺上皮细胞中NFκB活性后,正常间质细胞内MMP2/MMP9表达和分泌仍可被诱导增强；而正常间质细胞中NFκB活性被阻断后,其细胞内MMP2/MMP9表达和分泌则未能被诱导增强。研究结论①显著性增强的PI3K/PTEN/AKT信号通路、IκB/NFκB信号通路以及MAPKs/ERK1/2信号通路活性存在于子宫内膜异位症在位及异位腺上皮细胞；②PTEN蛋白及mRNA表达缺失存在于子宫内膜异位症在位及异位内膜腺上皮细胞;在体外,17β雌二醇可以通过IκB/NFκB信号通路下调原代分离培养的正常内膜及子宫内膜异位症在位及异位内膜腺上皮细胞中PTEN蛋白及mRNA的表达水平；③在体外,17β雌二醇可以通过PI3K/PTEN/AKT信号通路、IκB/NFκB信号通路以及MAPKs/ERK1/2信号通路促进正常内膜及子宫内膜异位症在位及异位内膜腺上皮细胞的生长增殖；④在正常内膜及子宫内膜异位症在位、异位内膜腺上皮细胞中,PI3K/PTEN/AKT信号通路与IκB/NFκB信号通路之间可以相互正相调节,且同时为MAPKs/ERK1/2信号通路的上游调节信号；⑤促细胞增殖性正反馈环：高雌激素水平→高PI3K/PTEN/AKT信号通路活性→高IκB/NFκB信号通路活性→低PTEN表达→高PI3K/PTEN/AKT信号通路活性,存在于子宫内膜异位症在位及异位内膜腺上皮细胞；⑥子宫内膜异位症在位及异位内膜腺上皮细胞中具有较正常腺上皮细胞较高的Survivin表达水平；⑦在正常及子宫内膜异位症在位内膜中,间质细胞可通过PI3K/PTEN/AKT/Survivin信号通路调节腺上皮细胞的生长增殖,而子宫内膜异位症异位内膜间质细胞部分失去了这种生长调节机制；⑧子宫内膜异位症在位及异位内膜腺上皮细胞中具有较正常腺上皮细胞较高的MMP2及MMP9表达和分泌水平；⑨子宫内膜异位症在位及异位腺上皮细胞通过IκB/NFκB信号通路诱导正常内膜间质细胞中MMP2/MMP9的表达及分泌。研究意义本研究明确了PI3K/PTEN/AKT/Survivin信号通路、IκB/NFκB信号通路以及MAPKs/ERK1/2信号通路在子宫内膜异位症中的部分作用机制,为子宫内膜异位症发生发展机制研究提供了新的思路,为该疾病诊断,评估提供了新的标记物,为该疾病治疗提供了新的靶点。更多还原

【Abstract】 Endometriosis, a common disease in women of reproductive age, is characterized by the presence of functional endometrium-like glands and stromal tissues in sites outside of the uterine cavity. Endometriosis is an estrogen-dependent chronic disease with chronic pelvic pain and infertility are the main clinical features. Many studies have been reported about this disease; however, the pathogenesis of it is still unclear. In the present research, via tissular and cellular study, we investigated the presence and role of PI3K/PTEN/AKT/Survivin signaling and IκB/NFκB signaling in endometriosis.Object①To determine the presences and activities of proliferative signaling pathways:PI3K/PTEN/AKT pathway and IκB/NFκB pathway in endometriotic ectopic and eutopic endometria;②To decide the effect of 17βestrodial on the proliferation of endometriotic epithelial cells, and the role of these pathways in this effect;③To investigate the role of these pathways in the stromal-epithelial cell cross-talk in endometriosis.Methods①Normal endometrium and eutopic/ectopic endometrium from patients with endometriosis were obtained during operation. The tissues were divided into two parts:one for formalin fixation and immunohistochemistry, one for primary cell isolation and culture;②Determining the expression and activity of PI3K/PTEN/AKT pathway and IκB/NFκB pathway in normal and endometriotic eutopic endometria using immunohistochemistry analysis;③Cell isolation and primary culture:sterile normal and endometriotic endometria were obtained during operation. Tissues were treated with 0.25% collagenase IA. Normal and endometriotic eutopic and ectopic stromal cells and epithelial cells were isolated and cultured in vitro. Stromal cells and epithelial cells were characterized by immunocytochemistry analysis using vimentin and keratin antibodies;④Determining AKT and ERKl/2 phosphorylation to decide the activities of PI3K/PTEN/AKT pathway and MAPKs/ERK1/2 pathway in normal and endometriotic epithelial cells using Western Blot analysis; determining the DNA-binding activity of NFκB to decide the activity of IκB/NFκB pathway using electrophoretic mobility shift assay (EMSA);⑤Establishing stromal cell-epithelial cell co-culture systems:mixing stromal cells and epithelial cells in a ratio of 1:1 directly to establish contact-co-culture system; using conditioned medium, cell lysates, and Millipore transwell system to establish non-contact-co-culture system; using contact and non-contact co-culture systems to establish normal stromal cell-epithelial cells co-culture system, endometriotic stromal cell-epithelial cell co-culture system, and normal stromal cell-endometriotic epithelial cell co-culture system, endometriotic stromal cell-normal epithelial cell co-culture system;⑥Detecting the expression and location of Survivin, a member of inhibitor of apoptosis (IAP) family, in normal and endometriotic eutopic endometria using immunohistochemistry analysis; determining the expression and subcellular location of Survivin in primarily cultured normal and endometriotic eutopic epithelial cells using immunocytochemistry analysis and Western Blot analysis;⑦Determining the expression and secretion of matrix metalloproteinases 2/9 (MMP2/9) and tissue inhibitors of metalloproteinases 1/2 (TIMP1/2) using Western Blot analysis; detecting the activity of MMP2/9 in cell culture supernatants using gelatinase zymography;⑧Deciding the proliferation of all kinds of cells under different treatments using MTT test.Results①Significantly higher AKT phosphorylation and lower PTEN expression were present in endometriotic eutopic endometria;③Significantly higher AKT and ERK1/2 phosphorylation, lower PTEN expression, and higher NFκB DNA-binding activity were present in primarily cultured endometriotic eutopic and ectopic epithelial cells; higher MMP2/9 expression and secretion were also present in primarily cultured endometriotic eutopic and ectopic epithelial cells, while only weak MMP2/9 expression and secretion were present in normal and endometriotic stromal cells;③17βestrodial significantly enhanced AKT and ERK phosphorylation, NFκB DNA-biding activity in normal and endometriotic epithelial cells; also,17βestrodial decreased PTEN protein and mRNA expression and promoted cell proliferation; specific PI3K/PTEN/AKT pathway inhibitor LY294002, specific MAPKs/ERK1/2 pathway inhibitor U0126, and specific IκB/NFκB inhibitor PDTC significantly inhibited the background and estrogen-stimulated AKT and ERK1/2 phosphorylation, NFκB DNA-binding activity in normal and endometriotic epithelial cells; and also decreased the background and estrogen-induced cell proliferation; meanwhile, PDTC significantly abolished the PTEN-decrease by 17βestrodial while LY294002 and U0126 could not; also, LY294002 significantly inhibited the background and estrogen-induced NFκB DNA-binding activity; in converse, IκB/NFκB pathway inhibitor PDTC significantly inhibited the background and estrogen-induced AKT phosphorylation; MAPKs/ERK1/2 pathway inhibitor U0126 could only weakly inhibit estrogen-induced AKT phosphorylation and NFκB DNA-binding activity; also, LY294002 and PDTC could only inhibit estrogen-induced ERK1/2 phosphorylation;④The expression of Survivin existed in normal and endometriotic epithelial cells, and endometriotic epithelial cells showed higher Survivin expression compared with normal epithelial cells; PI3K/PTEN/AKT pathway inhibitor LY294002 could significantly inhibit Survivin protein expression; in the co-culture system, conditioned medium by normal and endometriotic eutopic stromal cells could inhibit AKT phosphorylation, Survivin expression, and cell proliferation in normal and endometriotic eutopic epithelial cells, and progesterone could enhance this inhibition; conditioned medium by endometriotic ectopic stromal cells could not inhibit AKT phosphorylation, Survivin expression, and cell proliferation in normal or endometriotic ectopic epithelial cells; in vitro, the proliferation of epithelial cells from endometriotic eutopic endometria＞ectopic endometria＞normal endometria;⑤In the co-culture system, conditioned medium and cell lysates from endometriotic eutopic and ectopic epithelial cells could induce higher MMP2/9 expression and secretion in normal stromal cells; and when the activity of IκB/NFκB pathway in epithelial cells was inhibited using PDTC, the MMP2/9 expression and secretion in stromal cells could still be induced; however, when the activity of IκB/NFκB pathway in stromal cells was inhibited using PDTC, the MMP2/9 expression and secretion could not be induced.Conclusion ①Significantly higher activities of PI3K/PTEN/AKT pathway, IκB/NFκB pathway, and MAPKs/ERK1/2 pathway were present in endometriotic eutopic and ectopic epithelial cells;②Loss of PTEN protein and mRNA expression existed in endometriotic eutopic and ectopic epithelial cells; in vitro,17βestrodial could decrease PTEN protein and mRNA expression in normal and endometriotic eutopic and ectopic epithelial cells via IκB/NFκB pathway;③In vitro,170 estrodial could promote cells proliferation in normal and endometriotic eutopic and ectopic epithelial cells through PI3K/PTEN/AKT pathway, IκB/NFκB pathway, and MAPKs/ERK1/2 pathway;④In normal and endometriotic epithelial cells, PI3K/PTEN/AKTsignaling pathway and IκB/NFκB signaling pathway could be positively regulated by each other, and both of the pathways were upstream regulator of MAPKs/ERKl/2 signaling pathway;⑤Proliferative positive feedback loop:higher estrogen level→higher PI3K/PTEN/AKT pathway activity→higher IκB/NFκB pathway activity→decreased PTEN expression→higher PI3K/PTEN/AKT pathway activity, was present in endometriotic eutopic and ectopic epithelial cells;⑥Higher Survivin expression was present in endometriotic epithelial cells;⑦In normal and endometriotic ectopic endometria, stromal cells could regulated cell proliferation in epithelial cells through PI3K/PTEN/AKT/Survivin pathway, which was lost in endometriotic ectopic endometria;⑧Higher MMP2/9 expression and secretion were present in endmetriotic epithelial cells;⑨Endometriotic eutopic and ectopic epithelial cells induced MMP2/9 expression and secretion in normal stromal cells via IκB/NFκB pathway. In the present studies, we described the role of PI3K/PTEN/AKT pathway, IκB/NFκB pathway, and MAPKs/ERKl/2 pathway in endometriosis, which will bring new sight on studying the pathogenesis of this disease, will provide new marker for disease diagnosis and evaluation, and will provide new therapy target for the disease.更多还原