肥大细胞在子宫内膜异位症发病机理中作用的研究

发布时间：2018-03-04 21:00

本文选题：子宫内膜异位症　切入点：动物模型　出处：《浙江大学》2014年硕士论文　论文类型：学位论文

【摘要】：背景子宫内膜异位症(以下简称内异症)是指具有生长功能的子宫内膜组织(腺体和间质)出现在子宫腔被覆盖内膜及宫体肌层以外的其他部位的妇科常见疾病,严重影响妇女身心健康.疼痛(包括痛经、性交痛和慢性盆腔痛等)和不孕是内异症的主要临床症状,其患病率在育龄妇女中达10-15%。虽然内异症发病率呈现增高趋势,但其病因和发病机制以及相应内异症疼痛的发生机制至今仍然不清。近年大量研究发现,内异症病灶内出血刺激导致无菌性炎症,免疫炎症以及病灶神经异常生长导致中枢与外周神经敏化可能是内异症疼痛的主要发病机制。肥大细胞(Mast cells,MC)是免疫炎症反应中的重要细胞之一,一些学者研究发现MC脱颗粒与偏头痛、间质性膀胱炎、肠易激综合症、异位性皮炎等疾病的疼痛症状有明显的相关性。在神经病理性疼痛中,分布于神经和神经周围组织的MC即可脱颗粒释放类胰蛋白酶、肿瘤坏死因子(Tumor necrosis factor-α, TNF-α)、神经肽、P物质(Substance P, SP)、神经生长因子(Nerve growth factor, NGF)、细胞因子以及神经递质如乙酰胆碱等活性介质导致神经敏化,而敏化的神经可释放大量神经递质反过来激活MC促使其脱颗粒释放致痛物质,进一步促进神经敏化,从而导致神经病理性疼痛的持续状态。研究发现,内异症病灶存在MC数量增加以及其活性增强,且病灶中MC与神经纤维之间密切相关。内异症疼痛目前认为也是一种内脏和神经病理性疼痛,因而MC在内异症疼痛发生机制中发挥作用。研究表明,雌激素可结合MC膜上ER-a启动快速的钙离子流,促进人及小鼠MC活化脱颗粒释放相应的介质。色甘酸钠是MC膜稳定剂,能抑制其释放介质,其作用机制可能与MC78-kDa蛋白磷酸化有关。78-kDa蛋白由两段与MC膜突蛋白同源的染色体组成,在细胞表面与细胞内构架之间通过调节性功能的连接而参与信号传导,一旦膜突蛋白在磷酸化／脱磷酸化作用下结构发生改变,将导致细胞膜和细胞内部结构的位置重排,并可能使MC分泌颗粒。基于以上研究,我们设计了本实验,首先建立SD大鼠内异症模型,在此基础上我们给予MC脱颗粒促进剂(雌激素)及抑制剂(色甘酸钠)干预后,观察模型病灶大小变化以及与MC脱颗粒的关系,探讨MC在内异症发病中的作用,为临床内异症疼痛的治疗提供新思路。目的探讨肥大细胞在子宫内膜异位症发病机理中的作用。材料和方法取健康雌性未孕SD大鼠60只,采用自体子宫内膜移植法建立大鼠腹壁内异症模型,分两部分进行：第一部分(每组8只)大鼠造模后即每天臀部肌肉注射不同剂量雌激素(高剂量组：造模时同时切除双侧卵巢后给予外源性雌激素200μg/kg,低剂量组：造模时同时切除双侧卵巢后给予外源性雌激素100μg/kg,单纯造模组：造模时不切除双侧卵巢不给予外源性雌激素),两周后观察其对大鼠内异症病灶的大小,组织形态的影响,每组处死4只大鼠取材,剩余大鼠继续注射相应剂量雌激素两周,取材,比较病灶MC总数及其脱颗粒数、类胰蛋白酶、NGF的表达,血清TNF-α、雌二醇(Estradiol, E2)水平；第二部分(分别10只、10只、8只、8只)大鼠造模两周后观察大鼠内异症病灶的大小,随后每天腹腔注射不同剂量色甘酸钠(高剂量组20mg/kg,低剂量组10mg/kg,溶剂组,空白对照组),连续用药两周处死取材,检测指标同上。统计方法 SPSS20.0软件进行分析,结果以X±S表示,进行正态性和方差齐性检验,多组间数据比较用单因素方差分析,两组间数据比较采用t检验,检验水准取0.05. 结果 1.雌激素干预组两周和四周时血清E2浓度均显著高于单纯造模组(P0.05),四周时高剂量雌激素干预组血清TNF-a浓度显著高于单纯造模组(P0.05)。 2.雌激素干预组两周和四周病灶体积显著大于单纯造模组(P0.05)。 3.无论是两周还是四周,低剂量雌激素干预组甲苯胺蓝染色脱颗粒／总的MC数比值显著高于单纯造模组(P0.05)。 4.雌激素干预组与单纯造模组间病灶类胰蛋白酶的表达未见明显差异(P0.05),四周时高剂量雌激素组NGF的表达显著高于单纯造模组(P0.05)。 5.色甘酸钠治疗组与溶剂组、空白对照组间血清E2水平无明显差异(P0.05),色甘酸钠高剂量治疗组血清TNF-a浓度显著低于溶剂组和空白对照组(P0.05)。 6.色甘酸钠干预组两周和四周时病灶体积与溶剂组、空白对照组均无明显差异(P0.05)。 7.色甘酸钠高剂量治疗组甲苯胺蓝染色活化的MC数显著显著低于溶剂组(P0.05),脱颗粒／总的MC数比值显著低于溶剂组和空白对照组(P0.05)。 8.色甘酸钠高剂量治疗组类胰蛋白酶表达显著低于溶剂组和空白对照组(P0.05),色甘酸钠治疗组NGF的表达略低于溶剂组和空白对照组,但统计学上无明显差异(P0.05)。结论 1.雌激素可促进内异病灶的生长,可通过激活肥大细胞,促使其脱颗粒释放TNF-α、NGF介导内异症的发病。 2.色甘酸钠可通过稳定肥大细胞抑制其脱颗粒,减少TNF-α、类胰蛋白酶的释放,缓解内异症症状。
[Abstract]:background
Endometriosis (hereinafter referred to as EMS) is a function of the growth of endometrial tissue (glands and stroma) of common gynecological diseases occur in other parts of the uterine cavity is covered outside the endometrium and uterine muscle layer, seriously affecting the health of women. The pain (including dysmenorrhea, chronic pelvic pain and dyspareunia) and infertility is the main clinical symptoms of endometriosis, and its prevalence in women of childbearing age in 10-15%. although endometriosis incidence showed increasing trend, but its etiology and pathogenesis and mechanism of endometriosis pain is still unclear. In recent years, many studies found that endometriosis lesions bleeding stimulation leads to aseptic inflammation, immune inflammation and nerve lesions lead to abnormal growth in central and peripheral nerve sensitization may be the main pathogenesis of endometriosis pain.
Mast cells (Mast cells MC) is one of the important cells in the immune inflammatory reaction, some scholars found that MC degranulation and migraine, interstitial cystitis, irritable bowel syndrome, there is a significant correlation between the symptoms of atopic dermatitis and other diseases. In neuropathic pain, peripheral nerve and nerve distribution in tissue MC can degranulation tryptase release, tumor necrosis factor (Tumor necrosis factor- TNF- alpha, alpha), neuropeptide, substance P (Substance P SP), nerve growth factor (Nerve growth, factor, NGF), cytokines and neurotransmitters such as acetylcholine activity medium causes sensitization, and sensitization of nerve the MC in turn activates the degranulation to release pain caused by substances releasing large amounts of neurotransmitters, further promote nerve sensitization, leading to persistent neuropathic pain. The study found that endometriosis lesions M The number of C increased and its activity increased, and MC in lesions was closely related to nerve fibers. The pain of endometriosis is also considered as a visceral and neuropathic pain. Therefore, MC plays a role in the pathogenesis of pain in endometriosis.
Research shows that estrogen can be combined with the MC film on the ER-a promoter calcium fast flow, promote human and mouse MC activation and degranulation to release the corresponding medium. Cromolyn sodium is MC membrane stabilizer, could inhibit the release of mediators, and its mechanism may be related to MC78-kDa protein phosphorylation of.78-kDa protein is composed of two sections and chromosome MC moesin homology, between the cell surface and intracellular regulatory framework by connecting the function in signal transduction, once the moesin structural changes in phosphorylation / dephosphorylation, will cause the position of rearrangement of the cell membrane and the structure, and may make the MC secretory granules.
Based on the above research, we designed this experiment, we establish the SD rat model of endometriosis, on this basis, we give the degranulation of MC promoter (estrogen) and inhibitor (sodium cromoglycate) intervention, observation model of lesion size change and the relation with MC degranulation, discuss MC in the pathogenesis of endometriosis the role, to provide new ideas for clinical treatment of endometriosis pain.
objective
To investigate the role of mast cells in the pathogenesis of endometriosis.
Materials and methods
60 healthy female non pregnant SD rats to establish rat abdominal wall endometriosis model by autologous transplantation of endometrium was divided into two parts: the first part (n = 8) rats after every intramuscular injection of different dose of estrogen (high dose group: Modeling and removing the ovaries after the exogenous estrogen 200 g/kg, low dose group, at the same time after ovariectomy exogenous estrogen 100 g/kg, simple model: the model without ovariectomy not exogenous estrogen), observing the rat endometriosis lesion size after two weeks, the morphology each group, 4 rats were killed at the remaining rats injected corresponding dose of estrogen to two weeks, were compared and total number of lesions in MC degranulation, tryptase, NGF expression, serum TNF-, estradiol (Estradiol, E2) level; the second part ( Respectively 10, 10, 8, 8) were observed in rats of endometriosis lesion size in rats after two weeks, followed by intraperitoneal injection of different doses of color acid sodium (20mg/kg of high dose group, low dose group 10mg/kg, solvent group, blank control group), two weeks were sacrificed the detection index, ibid.
statistical method
SPSS20.0 software was used to analyze the results. The results were expressed by X + S. Normality and homogeneity of variance were tested. The data of multiple groups were compared by one-way ANOVA. The data between two groups were compared with t test, and the test level was 0.05..
Result
1. the serum E2 concentration in the estrogen intervention group was significantly higher than that in the simple model group (P0.05) at two weeks and 4 weeks, and the serum TNF-a concentration in the high dose estrogen intervention group was significantly higher than that in the simple model group (P0.05).
2. the volume of the lesion in the two week and four weeks of the estrogen intervention group was significantly greater than that of the simple model group (P0.05).
3. either two weeks or around, low dose estrogen intervention group with toluidine blue staining degranulation / total number of MC was significantly higher than those in the model group (P0.05).
4. there was no significant difference in the expression of tryptase between the estrogen intervention group and the simple model group (P0.05), and the expression of NGF in the high dose estrogen group was significantly higher than that in the simple model group (P0.05).
There was no significant difference in serum E2 level between the 5. color sodium glycyrrhizinate treatment group and the solvent group and blank control group (P0.05). The serum TNF-a concentration in the high-dose sodium glycyrrhizinate group was significantly lower than that in the solvent group and the blank control group (P0.05).
There was no significant difference between the focus volume and the solvent group at two weeks and four weeks in the 6. color sodium glycolate intervention group, and there was no significant difference between the control group (P0.05).
7. color display MC cromoglycate high dose treatment group was significantly lower than that with toluidine blue staining activated solvent group (P0.05), MC ratio was significantly lower than the total degranulation / solvent group and blank control group (P0.05).
Tryptase expression in high dose 8. sodium glycolate group was significantly lower than that in the solvent group and blank control group (P0.05). The expression of NGF in the sodium glycyrrhizinate treatment group was slightly lower than that in the solvent group and blank control group, but there was no significant difference between them (P0.05).
conclusion
1. estrogen can promote the growth of endometriosis, and can induce the release of TNF- a by activating mast cells, and NGF mediates the pathogenesis of endometriosis.
2. sodium glycine can inhibit the degranulation by stabilizing the mast cells, reduce the release of TNF- - A, trypsin like, and relieve the symptoms of endometriosis.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.71

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