研究背景 子宫内膜异位症(Endometriosis，EMs)是一种常见的妇科炎症性疾病，其特征是子宫内膜细胞种植于宫腔被覆粘膜外的其它部位。EMs好发于6～10％育龄期女性，主要症状为疼痛和不孕。超过80％的EMs患者表现为慢性疼痛，如痛经、腹痛、性交痛、非月经相关慢性盆腔痛等。疼痛是EMs最常见和最严重的症状之一，严重影响女性生活质量、社会关系和心理健康。近年来，大量动物模型试验和临床数据发现，EMs可刺激炎症微环境形成，新生神经纤维生成、感觉传入神经纤维比例增加等，为解释EMs引起的各种疼痛问题提供多种可能的理论，然而EMs疼痛发生发展的确切机制至今尚不清楚。EMs疼痛的致病因素是多元且复杂的，其发病机制是多个因素相互作用，而非单一因素的单独作用。因此，对炎症、外周神经纤维与中枢神经系统(central nervous system，CNS)间相互作用机制深入研究，有助于充分认知和理解EMs疼痛。 近期研究表明，EMs疼痛是一种神经病理性疼痛。异位内膜组织募集大量巨噬细胞、肥大细胞、中性粒细胞等，释放多种炎症和(或)细胞因子形成炎症微环境。巨噬细胞是EMs发生发展的关键细胞，参与多种炎症因子和细胞因子的分泌，其中大多数分子被证明与疼痛发生具有相关性。此外，异位病灶中新生神经血管生成，不仅加剧局部炎症，而且为炎症微环境与神经系统相互作用创造条件。炎症因子和神经肽能够直接刺激或使感觉神经纤维敏感化，兴奋伤害感受性神经元，导致痛觉超敏和(或)痛觉过敏。长期暴露于炎症环境的感觉传入神经不仅发生外周神经敏化，最终也会导致中枢神经敏化，形成慢性持续性疼痛，例如很多EMs患者表现为非雌激素依赖性的慢性持续性盆腔痛。而这种类型的疼痛可能与疾病程度、类型及外界条件变化没有相关性。但EMs炎症与神经系统相互作用的调控机制却鲜有报道。 神经病理性疼痛中枢敏化最重要的指标是小胶质细胞活化。一旦中枢神经受到外周伤害性刺激后，小胶质细胞首先被激活并维持很长时间。小胶质细胞活化与很多细胞因子相关，其中Fractalkine(FKN)是CX3C-类趋化因子，通过结合FKN唯一受体CX3CR1，作用于表达CX3CR1的效应细胞发挥生物学功能。FKN广泛分布于外周组织和CNS，包含两种形式：膜结合型和分泌型。在外周组织中，FKN主要表达于内皮细胞和上皮细胞。膜结合型FKN主要作为黏附分子，促进细胞间粘结和相互作用；分泌型FKN具有趋化作用，招募大量炎症细胞迁移至病灶周围。在CNS中，表达FKN的细胞局限于神经元，而CX3CR1只表达于小胶质细胞。虽然研究报道EMs患者腹腔液和病灶中FKN显著增多，但FKN／CX3CR1信号通路在EMs疼痛中的作用机制仍不清楚。 许多盆腹腔EMs临床病例显示异位病灶能够直接侵犯或影响神经(如坐骨神经、阴部神经、股神经、闭孔神经)和(或)脊神经根。但因盆腹腔EMs动物模型的局限性和获取临床EMs患者神经系统标本存在困难，使得研究EMs炎症与神经系统相互作用成为非常棘手和极具挑战性的难题。因此，本研究采用先前发表的坐骨神经EMs大鼠模型，旨在探究FKN／CX3CR1信号通路促进EMs疼痛发生的作用机制。 第一部分 坐骨神经子宫内膜异位症大鼠模型的建立 研究目的： 1.探讨坐骨神经EMs大鼠模型的构建方法和技术要点，为应用大鼠模型研究EMs炎症和神经系统相互作用的理论依据奠定基础。 2.评估坐骨神经EMs大鼠模型疼痛行为学变化和临床模拟程度，说明该模型用于EMs疼痛机制研究的可行性。 3.根据疼痛行为学曲线，判断坐骨神经EMs大鼠模型疼痛状态稳定建立的时间点，筛选检测分子生物学变化的最优时间。 材料及方法： 无特定病原体(Specific Pathogen Free，SPF)级雌性SD(Sprague Dawley)大鼠24只，约200-280g，构建坐骨神经EMs大鼠模型。造模手术前，应用电子Von Frey纤维试验、棉拭子试验和丙酮蒸发试验检测疼痛行为学基线(baseline)，然后随机分为3组：内膜移植组、脂肪移植组、正常对照组，8只／组。内膜移植组采用自体子宫内膜移植环绕股骨中上段坐骨神经，即坐骨神经EMs大鼠模型：脂肪移植组采用自体子宫周围脂肪组织；正常对照组不做任何处理。在术后第1， 3，5，7，14，21，28天，应用电子Von Frey纤维试验、棉拭子试验和丙酮蒸发试验分别检测手术侧与未手术侧后肢的疼痛行为学变化；绘制疼痛行为学曲线，根据曲线判断造模成功的最早和最优时间点，用于后续分子生物学验证。 基于疼痛行为学曲线判定最优时间点——术后第21天，选取48只SPF级SD大鼠，分为上述3组，16只／组，取材前检测机械性痛阈再随机分为2组，分别用于Western blot和石蜡包埋，8只／组。应用解剖显微镜观察术后第21天移植组织的在体和大体特征。应用苏木素伊红染色(H&E染色)观察术后第21天移植组织的病理特征，2名妇科病理专家判断术后第21天坐骨神经EMs大鼠模型是否成功建立。剩余的蛋白组织和蜡块用于第二部分机制研究。 研究结果： 1.疼痛行为学结果 各组大鼠所有疼痛行为学试验的基线一致。电子Von Frey纤维试验结果显示：术后第3天起，内膜移植组手术侧后肢表现为明显的机械性痛觉过敏，直至实验结束均明显高于其他组(P<0.05)。棉拭子试验结果显示：从术后第5至28天，内膜移植组手术侧后肢足底表现出对良性刺激明显的机械性痛觉超敏(即触觉诱发痛)(P<0.01)。丙酮蒸发试验结果显示：术后第7天起，内膜移植组手术侧后肢足底表现出明显的冷痛觉超敏，直至第28天均明显高于其他组(P<0.001)。但脂肪移植组和正常对照组手术侧后肢无明显痛觉过敏和超敏；各组大鼠未手术侧后肢的痛觉水平基本无变化(P>0.05)。疼痛行为学曲线显示：痛觉敏感性于术后第21天达到顶峰，提示第21天是坐骨神经EMs大鼠模型疼痛状态稳定建立的最优时间点。 2.大体和病理特征 大体观察结果显示：在术后第21天，内膜移植组子宫内膜包绕坐骨神经形成椭圆形囊胞，直径约6±1.18㎜，脂肪移植组没有形态变化。H&E染色结果显示：内膜移植组异位病灶可观察到典型子宫内膜腺上皮细胞，在异位内膜间质和坐骨神经周围充满大量炎症细胞，尤其是含铁血黄素细胞，与人EMs病理特征一致。脂肪移植组和正常对照组手术侧与各组未手术侧坐骨神经均没有明显的炎症细胞浸润现象。 研究结论： 1.坐骨神经EMs能够引起痛觉过敏和痛觉超敏，具有人EMs典型的大体和病理特征，在一定程度上能够模拟临床疾病。 2.坐骨神经EMs能够诱导大量炎症细胞浸润至神经束内，为研究炎症和神经系统相互作用提供了基础。 关键词：子宫内膜异位症；动物模型；疼痛；炎症；神经 第二部分 Fractalkine／Cx3CR1在子宫内膜异位症疼痛外周敏化中潜在机制的研究 研究目的： 1.检测FKN／CX3CR1和巨噬细胞在异位子宫内膜、正常子宫内膜和各组坐骨神经中的定位和表达情况。 2.分析FKN／CX3CR1在坐骨神经内的表达与EMs疼痛程度的相关关系。 3.探讨FKN／CX3CR1信号通路在EMs炎症与外周神经相互作用中的潜在机制。 材料及方法： 采用第一部分研究剩余的蛋白组织和蜡块用于第二部分机制研究。应用免疫组化检测大鼠异位子宫内膜组织，正常子宫内膜组织、各组大鼠手术侧坐骨神经中FKN／CX3CR1和Iba1(活化小胶质细胞／巨噬细胞标记分子)的定位和表达。应用非参数秩和检验分析坐骨神经中FKN／CX3CR1表达与EMs疼痛程度的Kendall和Spearman相关系数。应用Western blot检测移植组织内FKN／CX3CR1蛋白表达，区分分泌型和膜结合型FKN。应用免疫荧光共定位检测FKN和lba1、 CX3CR1和PGP9.5(神经纤维标记分子)、CX3CR1和MBP(神经纤维髓鞘标记分子)的共定位及表达。应用多色免疫荧光检测FKN、CX3CR1、MBP、Iba1和DAPI的共定位和表达。 研究结果： 1.FKN／CX3CR1和巨噬细胞在组织中的表达情况： 免疫组化结果显示：与大鼠正常子宫内膜组织相比，异位子宫内膜组织中FKN／CX3CR1表达显著增高，巨噬细胞浸润增加(P<0.01)；内膜移植组坐骨神经中FKN(P<0.001)、CX3CR1(P<0.01)和Iba1表达(即巨噬细胞数，P<0.001)均明显高于脂肪移植组和正常对照组。Western blot结果显示：内膜移植组中FKN／CX3CR1和Iba1蛋白表达量均显著高于脂肪移植组和正常对照组(所有P<0.01)；内膜移植组中膜结合型FKN明显多于分泌型FKN。 2.FKN／CX3CR1表达与EMs疼痛程度的相关关系： 非参数秩和检验结果显示：坐骨神经中FKN／CX3CR1表达与机械性痛阈呈负相关关系，因此与疼痛严重程度呈正相关关系。 3.FKN／CX3CR1信号通路在EMs炎症和神经纤维间潜在的作用机制： 免疫荧光共定位结果显示：内膜移植组坐骨神经中FKN阳性的活化巨噬细胞数和CX3CR1阳性的有髓神经纤维数明显多于脂肪移植组和正常对照组(P<0.001)。多色免疫荧光结果显示：内膜移植组坐骨神经中FKN阳性的活化巨噬细胞与CX3CR1阳性的有髓神经纤维表达部位重叠，且FKN，Iba1，CX3CR1， MBP，DAPI均阳性的数量明显高于移植脂肪组(P<0.05)和正常对照组(P<0.01)。 研究结论： 1.EMs导致神经纤维中FKN／CX3CR1表达增加和大量巨噬细胞浸润，且FKN／CX3CR1表达与EMs疼痛严重程度呈正相关，说明FKN／CX3CR1信号通路可能参与炎症与神经纤维相互作用促进EMs疼痛外周敏化发生。 2.EMs病灶神经纤维内的巨噬细胞通过表达FKN与神经纤维髓鞘表面的CX3CR1配受体结合，促进巨噬细胞与神经纤维相互作用介导EMs外周神经敏化的发生发展。 关键词：子宫内膜异位症；巨噬细胞；外周敏化；FKN；CX3CR1 第三部分 Fractalline／CX3CR1在子宫内膜异位症疼痛传导和中枢敏化中作用机制的研究 研究目的： 1.探讨背根神经节(dorsal root ganglia，DRG)FKN／CX3CR1在EMs疼痛传导中的潜在作用。 2.研究脊髓FKN／CX3CR1在EMs疼痛中枢敏化的作用机制。 材料及方法： 采用第一部分研究获取的L4-L6脊髓蜡块和蛋白组织(内膜移植组，脂肪移植组和正常对照组)。应用免疫组化、免疫荧光和Western blot初步检测EMs与脊髓FKN／CX3CR1／P38-MAPK和小胶质细胞总数的相关性。 SPF级雌性SD大鼠132只，约200-280g，行鞘内置管手术，观察1周，剔除精神系统紊乱等大鼠，余下的120只大鼠随机分为5组：正常对照组，内膜移植+鞘内注射lgG组，脂肪移植+鞘内注射IgG组，内膜移植+鞘内注射FKN中和抗体组，脂肪移植+鞘内注射FKN中和抗体组，24只／组。每组大鼠分别随机分配用于疼痛行为学试验(n=8)，Western blot(n=8)和石蜡包埋(n=8)。术后第3，7，14，21天，鞘内给予FKN中和抗体和IgG。应用疼痛行为学试验检测术后1，3，5，7，14，21的长期疼痛行为学和电子Von Frey纤维试验检测鞘内注射后0h，0.5h，1h，3h的短期疼痛行为学。术后第21天取材前验证各组大鼠疼痛行为学。应用免疫组化和mRNA原位杂交分别检测L4-L6 DRG中FKN／CX3CR1的表达和定位情况。应用Western blot检测L4-L6脊髓FKN／CX3CR1、P38-MAPK、pP38-MAPK的蛋白表达。应用免疫荧光检测L4-L6脊髓灰质后角FKN和NeuN(神经元标记分子)、CX3CR1和Iba1、pP38-MAPK和Iba1的共定位和表达。 研究结果： 1.初步验证EMs与脊髓FKN／Cx3CR1／P38-MAPK／小胶质细胞的相关性 免疫组化和免疫荧光结果显示：与脂肪移植组和正常对照组相比，内膜移植组中脊髓灰质后角FKN／CX3CR1表达和小胶质细胞总数(OX42，静息和活动小胶质细胞标记分子)明显表达增加，但两对照组间无统计学意义(P>0.05)。 Western blot结果显示：内膜移植组脊髓P38-MAPK中蛋白磷酸化水平明显高于脂肪移植组(P<0.05)和正常对照组(P<0.01)，但两对照组间无统计学意义(P>0.05)。 2.疼痛行为学结果 长期疼痛行为学结果显示：从术后第7天至21天，鞘内给予FKN中和抗体明显减轻EMs大鼠的痛觉过敏和痛觉超敏。短期疼痛行为学结果显示：在术后第3，7，14，21天，鞘内给予FKN中和抗体后，EMs大鼠机械性痛阈逐渐回归基线水平，且在第7，14，21天，各组机械性痛阈在注射后3h内基本无变化。单次给予FKN中和抗体对缓解疼痛发挥长效作用。 3.DRG中FKN／CX3CR1的表达情况 免疫组化染色结果显示：与内膜移植+鞘内注射IgG组相比，鞘内注射FKN中和抗体明显减少DRG神经元内FKN表达；分泌型FKN只存在于内膜移植+鞘内注射IgG组DRG神经元周围神经纤维间质。CX3CR1mRNA原位杂交结果显示：鞘内注射FKN中和抗体抑制DRG神经元周围胶质细胞CX3CR1 mRNA的表达。 4.FKN／CX3CR1中枢敏化机制结果 Western blot结果显示：鞘内注射FKN中和抗体后，显著降低了脊髓中FKN／CX3CR1表达和P38-MAPK磷酸化水平。免疫荧光染色结果显示：脊髓灰质后角FKN主要位于神经元；抑制脊髓FKN表达后，脊髓灰质后角中CX3CR1和pP38-MAPK阳性的活化小胶质细胞数明显减少。 研究结论： 1.DRG和脊髓中FKN／CX3CR1表达与EMs疼痛中枢敏化相关，鞘内抑制FKN能减少活化小胶质细胞，缓解痛觉过敏和痛觉超敏，甚至逆转疼痛发生。 2.FKN／CX3CR1信号通路可能促进DRG神经元.胶质细胞相互作用参与EMs疼痛传导。 3.脊髓FKN／CX3CR1／P38-MAPK信号通路可能参与EMs疼痛中枢敏化发生发展通过神经元-小胶质细胞相互作用。 关键词：子宫内膜异位症；疼痛；中枢敏化；FKN；CX3CR1

 Background Endometriosis (EMs) is a common chronic inflammatory gynecologic disorder that is characterized by the growth of endometrial epithelial and stromal cells outside the uterus. It affects 6%-10% of women of reproductive age. The common symptoms of EMs are pain and subfertility. More than 80% of EMs patients suffer from chronic pain. Pain symptoms associated with endometriosis include dysmenorrhea, dyspareunia, dysuria, abdominal pain and non-menstruation-related chronic pelvic pain. As one of the most common and serious symptoms of EMs, chronic pain profoundly affects women’s quality of life, social relationship and mental health. Recently, increasing evidence of animal model experiments and clinical data suggested that ectopic endometrium growth could induce inflammatory microenvironment and increase neuroangiogenesis/sensory nerve fibers and so on, which probably provide various potential theoretical basis for explaining the multifarious pain problems. However, the pathogenesis of pain associated with EMs is still unclear. The pathogenic factors of endometriotic pain are known to be multivariate and complex, so its occurrence and development is due to the multiple factors interaction rather than the single effect. In-depth study on the interaction of inflammation, peripheral nerve fibers and central nervous system will contribute to the fully recognizing and understanding the potential mechanism of endometriotic pain. Recent research data indicates that endometriotic pain is a type of inflammatory and neuropathic pain. During ectopic endometrial growth, large numbers of inflammatory cells like macrophages, mastocytes, and neutrophils are recruited to secrete a variety of pro-inflammatory/inflammatory cytokines and contributed to the inflammation microenvironment. Macrophages are the major source of pro-inflammatory cytokines/ chemokines, which play a role in the development of EMs. The majorities of these molecules have been demonstrated to be associated with the development of pain. Endometriotic lesions also exhibit increased neuroangiogenesis, which could be attributed to localized inflammation, and promote the interaction between inflammatory microenvironment and nerve fibers. While, inflammatory molecules and neuropeptides might directly activate or sensitize sprouting sensory fibers in the tissues or excite nociceptive neurons, which resulted in pain and/or mechanical hypersensitivity/ allodynia. Prolonged exposure of sensory neurons to inflammatory mediators would then lead to central sensitization and cause estrogen-independent pain, as observed in some patients with endometriosis and persistent pelvic pain. The level of pain may not be associated with severity, implant position or surrounding environmental conditions of EMs. However, the concrete mechanism of cytokines/ chemokines directly function on nerve fibers in endometriotic pain is rarely reported. Spinal microglia were thought to be the indicators of central sensitization in neuropathic pain. They were the first to become activated by peripheral nociceptive signals, and could remain active for several weeks. Cytokines—including fractalkine (FKN)—were thought to be associated with microglia activation. FKN is a member of the chemokine family that consists principally of secreted proinflammatory molecules, and acts by binding to a unique receptor, CX3CR1, expressed on corresponding cells. The chemokines widely expressed in the periphery and central nervous system (CNS) have two forms: a membrane-bound protein and soluble forms (sFKN), each of which mediates different biologic functions. In the periphery, predominant FKN-expressing cells are endothelial and epithelial cells. Membrane-bound FKN serves as an adhesion molecule to promote adhesion of inflammatory cells, while sFKN is a potent chemoattractant for the recruitment of inflammatory cells during chronic inflammation. In the CNS, FKN-expressing cells are restricted to the intrinsic neurons and sensory afferents, whereas the FKN receptor (CX3CR1) is expressed exclusively in the spinal microglia. Although increased levels of FKN were found in the peritoneal fluid of women with endometriosis, the mechanism of FKN/CX3CR1 signaling pathway on endometriotic pain remains unclear. Many clinical cases of endometriosis in the abdominopelvic cavity were reported to directly affect nerves (including the pudendal, obturator, femoral, and sciatic) or spinal roots. Due to the limitations of general endometriosis animal models and sampling nerve system from clinical EMs patients, it is extremely difficult and challenging to study the interaction mechanism between EMs inflammation and the nervous system., Herein we planned to explore the functional roles of FKN/CX3CR1 signaling pathway in contributing to endometriotic pain using a novel rat model of sciatic endometriosis that we originally described in a previous publication. PART I: Establishment of sciatic endometriosis rat model Objective: 1.To discuss the method and technical point of the sciatic EMs rat model, and provide a suitable model basis for the theoretical study on the interaction between inflammation and nervous system. 2.To evaluate pain behavior changes and discuss the feasibility of the sciatic EMs rat model on researching the mechanism of endometriotic pain. 3.According to pain behavior curve, we aimed to explore the optimal time point for the steady pain state and molecular biological changes in the sciatic EMs rat model. Materials & Methods: Female, 24, Sprague Dawley (SD) rats, Specific Pathogen Free (SPF), weighing 200-280g were used to establish the sciatic EMs rat model. Before modeling, pain behavior baseline of all the rats were detected by the electronic Von Frey test, tactile allodynia test and acetone test. Rats were randomly divided into 3 groups: endometrial graft group (Endo, n=8), fat graft group (Fat, n=8) and normal control group (Naive, n=8). In the endometrial graft group, the autologous endometrium was transplanted to surround the sciatic nerve in the middle and upper femoral—sciatic EMs rat model; in the fat graft group, the adipose tissue around the uterus was used to wrapping the sciatic nerve in the middle and upper femoral; the normal control group were without any treatment. On day 1,3,5,7,14,21,28 (post-operative day, POD) after transplantation, the electronic Von Frey test, tactile allodynia test and acetone test were used to monitor the pain behavior changes of the ipsilateral and contralateral hind paw, respectively. The pain behavior curve was used to estimate the earliest and optimal time point for the successful establishment of painful state, which was used for the verification of subsequent molecular biology. Based on the optimal time point determined by pain behavior curve - POD21, 48, SPF, SD female rats were selected and randomly allocated to the above 3 groups, 16 rats/ group, which were used to observe the general and pathological characteristics of the sciatic EMs rat model. Before collecting, all the rats were performed pain behavior test. Then, each group of rats was randomly divided into 2 groups, 8 rats/ group, which were prepared for Western blot and paraffin embedding, respectively. On POD21, the general features of graft tissues in vivo were evaluated with anatomic microscope. The histopathological characteristics of graft tissues were demonstrated using hematoxylin-eosin staining (H&E staining) to estimate whether the sciatic EMs rat model was successfully established on POD21. The remaining fresh tissues and paraffin blocks were used for the PART II and PART III. Results: 1.Pain behavior test The baseline threshold of rats in each group were kept identical. Electronic Von Frey test showed that rats with uterine graft developed significantly increased ipsilateral mechanical hypersensitivity since POD3, and kept continuously increasing compared to the contralateral hind paw and the control groups until the end of the experiment (P<0.05). Tactile allodynia test showed that rats in endometrial graft group developed significantly increased ipsilateral light touch-induced tactile allodynia from POD5 to POD28 compared to the contralateral hind paw and the control groups (P<0.01). Acetone test showed that, since POD7, rats with uterine graft developed significantly increased ipsilateral cold allodynia, and kept continuously increasing compared to the contralateral hind paw and the control groups until the end of the test (P<0.001). However, all the data showed that there was no statistically significant difference between the fat graft group and the normal control group (P>0.05). Also, there were no pain behavior changes in the contralateral hind paw of each group (P>0.05). The pain behavior curve showed that pain reached its peak on POD21, suggesting that POD21 was the optimal time for the steady painful state in the sciatic EMs rat model. 2.The general and histopathological features General observation results showed that ectopic endometrial tissue formed a single cyst wrapping around the sciatic nerve in the endometrium graft group on POD21, about 6± 1.18㎜ in diameter. The fat graft group had no morphological changes. H&E staining of the sciatic nerve cross-sections showed that uterine grafts exhibited typical glandular formations with columnar epithelial cells and hemosiderin-laden macrophages, accompanied by large numbers of inflammatory cells around the adjacent endometrial stroma and sciatic nerve, which were consistent with the pathological characteristics of human EMs. No inflammatory cells infiltration was observed in either the two control groups or contralateral hind paw. Conclusions: 1.The sciatic EMs could induce mechanical hyperalgesia and allodynia and have almost the same typical general and pathological characteristics as human EMs. To some extent, it could simulate clinical diseases. 2.The sciatic EMs could recruit large numbers of inflammatory cells to infiltrate into the nerve fiber bundles, which provides the foundation for studying the interaction between inflammation and nerve fibers. Key words: endometriosis; animal model; pain; inflammation; nerve fibers