【作者】 李斌；

【导师】 刘惜时；

【作者基本信息】 复旦大学 ， 妇产科学， 2011， 博士

【摘要】 子宫腺肌病是一种常见的妇科疾病,发病率介于5-70%之间且呈现逐年上升趋势,临床表现主要为痛经、子宫增大、不规则子宫出血及不育等。目前,子宫腺肌病的发病机制尚不清楚,其临床药物治疗效果不佳,手术治疗是目前最有效的治疗手段。子宫腺肌病已成为危害妇女健康的重要疾病之一。核因子κB (nuclear factor-kappa B, NF-κB)和激活蛋白-1(active protein-1, AP-1)通过促进炎症反应、抑制凋亡等过程参与多种疾病的病理发生。转录因子NF-κB和AP-1被认为在子宫内膜异位症的发病过程中可能起到关键作用。此外,研究显示其下游分子环氧化酶-2(cyclooxygenase-2, COX-2)、血管内皮生长因子(vascular endothelial growth factor, VEGF)和组织因子(tissue factor, TF)可能通过促进血管形成和炎症反应在子宫内膜异位症的发生和发展过程中发挥重要作用。但是,目前仅有的一些关于NF-κB和AP-1及其下游分子的研究几乎都集中在子宫内膜异位症上,子宫腺肌病的体内及体外相关研究尚无报道。目前,我们仍不清楚腺肌病病灶组织中NF-κB和AP-1的状态,是否为组成性激活?体外腺肌病细胞中异常表达的分子是否受到NF-κB和AP-1这些转录因子的调控?根据子宫内膜异位症中研究结果,我们推测在子宫腺肌症的发病过程中,当受到内外环境因素的刺激后,首先引起转录因子NF-κB及AP-1的异常激活,然后导致下游的炎症介质及一些细胞因子的异常表达,这些异常表达的分子最后促使了疾病的发生和发展,转录因子NF-κB及AP-1可能在整个疾病的发生和发展过程中起着中枢的作用。此外,有研究发现穿心莲内酯(andrographolide, Andro)能够特异性地下调NF-κB与DNA的结合能力,从而抑制其下游基因的表达。曲古霉素A (trichostatin A, TSA)能够抑制子宫内膜间质细胞的增殖和COX-2的表达以及小鼠内异症模型中异位病灶的生长。根据已有的研究结果,我们推测Andro和TSA很可能会通过影响转录因子NF-κB及AP-1的表达在子宫腺肌病中发挥作用。本课题将检测转录因子NF-κB和AP-1及其下游分子COX-2、VEGF和TF在子宫腺肌病病灶组织中的表达；研究体外腺肌病间质细胞中TNFa对转录因子NF-κB和AP-1的作用；研究Andro和TSA对腺肌病间质细胞中转录因子NF-κB和AP-1及其下游靶基因COX-2、VEGF和TF的作用。第一部分：转录因子NF-κB和AP-1及其下游分子在子宫腺肌病中的表达目的：检测子宫腺肌病病灶组织中转录因子NF-κB和AP-1以及下游分子COX-2、VEGF和TF的表达。方法：提取23例子宫腺肌病病灶组织和8例对照内膜组织中的细胞核蛋白和胞浆蛋白,EMSA方法检测核蛋白中转录因子NF-κB和AP-1与DNA的结合能力；Western Blot方法分别检测核蛋白和胞浆蛋白中NF-κB亚基P50和P65、AP-1亚基c-Jun和c-Fos及其下游分子COX-2、VEGF和TF的表达。结果：EMSA结果显示子宫腺肌病病灶组织中,转录因子NF-κB和AP-1为组成性激活,表达水平显著高于对照内膜组织(P<0.05),其中NF-κB在腺肌病组织中呈普遍性的组成性激活(87%),而AP-1在部分的腺肌病组织可检测到组成性激活(40%)。NF-κB的表达水平与腺肌病患者的痛经程度呈正相关(p<0.05)。Westren Blot结果显示腺肌病病灶组织中P50、P65、c-Jun、c-Fos、COX-2、VEGF以及TF的表达均显著高于正常对照内膜组(P<0.05)。结论：转录因子NF-κB和AP-1以及COX-2、VEGF和TF可能在子宫腺肌病的发病过程中发挥重要的作用。第二部分：TNFα对子宫腺肌病间质细胞中转录因子NF-κB和AP-1的作用目的：研究腺肌病间质细胞中TNFa对转录因子NF-κB和AP-1及其下游分子COX-2、VEGF和TF的作用,探讨子宫腺肌病中异常表达的分子是否受到转录因子NF-κB和AP-1的调控。方法：腺肌病间质细胞分别经TNFa以及NF-κB和AP-1的抑制剂处理后,提取细胞总蛋白,应用EMSA和Western Blot方法检测转录因子NF-κB和AP-1及其下游分子COX-2. VEGF和TF的表达。结果：在子宫腺肌病间质细胞中TNFa能够明显激活转录因子NF-κB和AP-1并上调下游分子COX-2、VEGF和TF的表达(P<0.05)；加入抑制剂后,COX-2、VEGF和TF的表达被降低,其中两种抑制剂联合使用后能够进一步降低三种分子的表达,抑制效果优于单一抑制剂的使用效果(P<0.05)。结论：腺肌病间质细胞中,转录因子NF-κB和AP-1参与了TNFa上调下游分子COX-2、VEGF和TF表达的过程。NF-κB和AP-1在子宫腺肌病的发病过程中可能起到中枢调控的作用第三部分：Andro和TSA通过干扰TNFa下调转录因子NF-κB和AP-1及其下游分子的表达目的：研究子宫腺肌病间质细胞中对转录因子NF-κB和AP-1及其下游分子COX-2、VEGF和TF的作用,为Andro和TSA在子宫腺肌病中的后续应用提供一定的理论指导。方法：子宫腺肌病间质细胞分别经Andro和TSA处理后,提取细胞核蛋白和总蛋白,应用EMSA和Western Blot方法检测细胞内NF-κB和AP-1及其下游分子COX-2、VEGF、TF的表达。结果：子宫腺肌病间质细胞中,Andro能够明显降低NF-κB与DNA的结合能力,下调COX-2、VEGF和TF的表达(P<0.05)。TSA能够持续性的下调TNFα诱导的COX-2和TF的表达,长时间的作用却显著上调TF表达(P<0.05)；TSA对NF-KB与DNA结合能力的作用呈现出了一种时间效应,即短时间内上调NF-KB与DNA的结合能力,而长时间的作用对NF-KB的DNA结合能力则起到下调作用；TSA能够持续性下调细胞内的P65和c-Jun的磷酸化水平(P<0.05),长时间处理后P50蛋白的表达会出现明显下调(P<0.05)；TSA能够持续下调JNK的磷酸化水平(P<0.05),而对P38的磷酸化无影响。TSA对ERK磷酸化水平的作用呈现短时间时下调,长时间时上调(P<0.05)；TSA联合使用Andro能够明显降低TSA单独使用时引起的TF表达上调(P<0.05),并能进一步下调COX-2和VEGF的表达(P<0.05)。结论：子宫腺肌病间质细胞内,Andro可以作为NF-KB抑制剂使用。TSA能够通过降低NF-KB和AP-1的转录能力而持续下调COX-2和VEGF的表达；TSA和Andro联合使用对下游分子COX-2、VEGF和TF的抑制效果优于TSA的单独使用。更多还原

【Abstract】 Adenomyosis is a common gynecologic disease and the etiology is poorly understood. The prevalence of adenomyosis reported in the literature ranges from5to70%. The common presenting symptoms are dysmenorrheal, enlarged tender uterus, subfertility, and abnormal uterine bleeding. About35%of women with adenomyosis are asymptomatic. Clinical treatment of it is a challenge, with hysterectomy being the treatment of choice. To date, adenomyosis already became one of the most dangerous threats to women health. Nuclear factor-kappa B (NF-kB) and active protein-1(AP-1) are involved in numerous pathologies and known to be a proinflammatory, antiapoptotic and mitogenic factor in many cell types. They are thought to play a pivotal role in pathogenesis of endometriosis. Recent researches indicated their target genes cyclooxygenase-2(COX-2), vascular endothelial growth factor (VEGF) and tissue factor (TF) might be involved in the pathogenesis of endometriosis through stimulating angiogenesis and inflammatory reaction. Published studies on NF-κB and AP-1and their target genes almost focused on the endometriosis, however, correlative studies on the adenomyosis has not been reported yet. To date, we still did not know the status of NF-κB and AP-1in adenomyotic lesions and whether over-expressed molecules were regulated by transcription factors NF-κB and AP-1in adenomyotic stromal cells. According to the researches in the endometriosis, we inferred that during the pathology of adenomyosis, transcription factors NF-κB and AP-1were firstly activated, and then gave rise to over-expresstion of inflammatory mediators and cytokines, and finally these abnormal molecules promoted the development of adenomyosis. Transcription factors NF-κB and AP-1might play a pivotal role in the development of the disease. In addition, it was found out that andrographolide (Andro) could specifically down-regulate NF-kB DNA binding activity and suppress the expression of target genes. Trichostatin A (TSA) could suppress the proliferation and expression of COX-2in the endometrial stromal cells and the growth of implanted lesions in the model mice with endometriosis. According to published results in the endometriosis, we inferred that Andro and TSA might play a role in the adenomyosis through affecting the expression of NF-kB and AP-1.In this study, we would detect the expression of transcription factors NF-kB and AP-1and their target genes COX-2, VEGF and TF in adenomyosis and the effect of TNFα on the transcription factors NF-κB and AP-1in the adenomyotic stromal cells in vitro, and the effect of Andro and TSA on transcription factors NF-κB and AP-1and their downstream target genes COX-2, VEGF and TF.Part I Expression of transcription factors NF-κB and AP-1and downstream molecules in adenomyosisObjective:To detect the expression of transcription factors NF-κB and AP-1and COX-2, VEGF and TF in adenomyotic lesions.Methods:Twenty-three adenomyotic lesion samples and8endometrial tissue samples from women with benign gynecological conditions, but not adenomyosis, were used to extract the nuclear and cytoplasm proteins, and their NF-κB and AP-1DNA binding activities were analyzed by electrophoretic mobility shift analysis (EMSA). Western blot analysis was used to evaluate the expression of NF-κB subunits p50and p65, AP-1subunits c-Jun and c-Fos, COX-2, VEGF and TF.Results:EMSA results showed that NF-κB and AP-1were constitutively activated in adenomyosis, and their activation levels were significantly higher than those of control group (P<0.05). In particular, NF-kB was universally activated constitutively in adenomyosis (87%), and while AP-1was constitutively activated in sizeable portion of adenomyotic lesions (40%). Besides, NF-kB activation level was positively correlated with the severity of dysmenorrheal in patients with adenomyosis (P<0.05). Western Blot results showed that the expressions of p50, p65, c-Jun, c-Fos, COX-2, VEGF and TF were all significantly higher than control endometrium (P<0.05).Conclusions:Transcriptional factors NF-κB and AP-1and COX-2, VEGF and TF may play a critical role in the pathogenesis of adenomyosis.Part Ⅱ The effect of TNFa on transcriptional factors NF-⒘B and AP-1in adenomyotic stromal cellsObjective:To investigate the effect of TNFa on the transcriptional factors NF-κB and AP-1and their downstream target genes COX-2, VEGF and TF in adenomyotic stromal cells and explore whether the over-expressed molecules are regulated by transcription factors NF-κB and AP-1in adenomyosis.Methods:Cultured adenomyotic stromal cells were treated with TNFa and inhibitors of NF-κB and AP-1, and EMSA and Western blot analysis were used to detect the expressions of NF-κB, AP-1and their target genes, COX-2, VEGF and TF.Results:In adenomyotic stromal cells, TNFa significantly induced further activation of NF-κB and AP-1, and increased the protein expression of COX-2, VEGF and TF (P<0.05). When inhibitors of NF-κB and AP-1were added, the expression levels of the three proteins were reduced, and were further reduced by the combined treatment of NF-κB and AP-1inhibitors (P<0.05).Conclusions:In adenomyotic stromal cells, transcriptional factors NF-κB and AP-1are involved in the up-regulation of TNFa-induced COX-2, VEGF and TF. Transcriptional factors NF-κB and AP-1may jointly play a pivotal role in the pathogenesis of adenomyosis Part III Andro and TSA reduce the TNFa-induced expression of NF-κB and AP-1and downstream molecules in adenomyotic stromal cellsObjective:To investigate the effect of Andro and TSA on transcriptional factors NF-κB and AP-1and their downstream target genes COX-2, VEGF and TF in adenomyotic stromal cells. The research may provide some new theoretical guidance for the further application of Andro and TSA.Methods:After adenomyotic stromal cells were treated with Andro and TSA respectively, the nucleoproteins and total proteins were prepared. EMSA and Western Blot analysis were used to detect the expression of TNFa-induced NF-κB and AP-1and their target genes COX-2, VEGF and TF, the three genes known to be involved in adenomyosis.Results:In adenomyotic stromal cells, Andro could suppress the NF-κB DNA binding activity, and significantly reduce the expression of COX-2, VEGF and TF (P<0.05). TSA could consistently reduce the protein products of TNFa-induced COX-2and TF (P<0.05), but long-playing treatment could significantly elevate the TF expression (P<0.05). Effect of TSA on NF-κB DNA binding activity was time-dependent: short-time treatment of TSA up-regulated NF-κB activity, but long-playing treatment down-regulate its activity. TSA could consistently reduce the phosphorylation of P65and c-Jun in adenomyotic stromal cells (P<0.05), and the expression of P50appeared observably down-regulation after24h treatment (P<0.05). Besides, TSA could reduce the phosphorylation of JNK (P<0.05), but the phosphorylation of P38had no change, and the phosphorylation of ERK showed that short-time treatment evidently down-regulated and long-playing treatment significantly up-regulated (P<0.05). When the combination of TSA and Andro was used to treat adenomyotic stromal cells, the protein products of COX-2, VEGF and TF were significantly suppressed (P<0.05), and the effect was superior to TSA treatmentκ(P<0.05). Conclusions:In adenomyotic stromal cells, Andro could be used as the NF-κB inhibitor. TSA could consistently down-regulate the expression of COX-2and VEGF through reducing the transcriptional activities of NF-κB and AP-1.Combined treatment of TSA and Andro was superior to that of TSA.更多还原