【作者】 李晓燕；

【导师】 冷金花； 樊庆泊；

【作者基本信息】 中国协和医科大学 ， 妇科学， 2009， 博士

【摘要】 疼痛是子宫内膜异位症(以下简称内异症)患者最突出、最典型的症状之一,其主要表现为月经期或经行前后出现的腹痛、腰骶部疼痛,甚至剧痛难忍,如此影响广大育龄妇女生活质量及身心健康的疾病,其疼痛机制尚不明确。近年来,对疼痛产生的机制进行了大量研究,最近研究较多的方向是子宫内膜异位病灶内的感觉神经纤维。研究认为,子宫内膜异位症的疼痛症状与病灶内感觉神经Aδ,感觉纤维C的分布异常增多相关。至于导致这种神经纤维分布增多现象的原因,尚缺乏相关报道。国外有报道内异症疼痛的神经解剖机制,但未对转换刺激的神经递质进行系统研究,国内对内异症疼痛机制研究目前还是空白。与痛觉感受有关的致痛物质主要是神经生长因子(nerve growth facter,NGF)、白介素及肿瘤坏死因子α等。NGF属于神经营养因子家族,是促进神经元存活的一类蛋白。近年来发现NGF与其受体结合后诱导自磷酸化作用,引发多种生物效应,其中包括诱导肥大细胞释放5-羟色胺,导致痛觉过敏,在调节神经病理性和非神经病理性疼痛中有着重要的作用。不同类型的内异灶与疼痛的关系不同,其中卵巢内膜异位囊肿与疼痛关系不明显,深部侵润型异位灶与痛经、CPP、性交痛以及大便痛都有密切的关系。2005年SCIENCE杂志载文认为内异症疼痛以及对雌激素敏感的机制之一就是异位内膜灶中神经支配的形成,从而对中枢神经元的活性产生广泛和多种的影响。但这些国外研究样本量均不大,研究分组缺乏不同程度疼痛症状之间,以及不同类型内膜异位灶之间的对比,其所得的结论尚未完全统一,还没有深入探讨神经纤维、神经递质以及致痛物质在内异灶中分布的量和质的改变和疼痛程度之间的相关性,并且没有对神经纤维的异常分布进行药物的干预研究。国内目前还没有开展此类的临床基础研究。研究目的1探讨不同类型的内异症病灶中NGF及其受体TrkA、P75的分布和表达的差异,以及与疼痛程度的关系;2探讨内异症痛经患者的在位内膜中是否存在神经生长因子及受体TrkA、P75异常的高表达并进行对比分析,进一步丰富“在位内膜决定论”;3探讨雌、孕激素及GnRHa对体外培养痛经患者在位内膜及正常内膜表达NGF、TrkA及P75NTR的影响。研究方法1免疫组化及免疫荧光双标法检测NGF、TrkA及P75在子宫内膜异位症患者病灶以及在位膜中的表达及共表达;2 Western Blot及RT-PCR法检测NGF、TrkA及P75在子宫内膜异位症患者病灶以及在位膜中的表达;3 Real-timePCR法检测NGF、TrkA及P75在子宫内膜异位症患者病灶以及在位膜中的表达;4体外培养在位内膜及正常内膜,并细胞免疫荧光、流式细胞技术以及RT-PCR法检测NGF、TrkA及P75在子宫内膜异位症患者病灶以及在位膜中的表达;5以不同浓度的雌、孕激素及GnRHa作用于体外培养在位内膜及正常内膜细胞,RT-PCR法检测NGF、TrkA及P75在细胞水平的表达;6同一浓度雌激素作用下,RT-PCR法检测不同时间点NGF、TrkA及P75在细胞水平的表达;7 ElISA法检测子宫内膜异位症患者血清中NGF以及痛觉敏感相关肽(孤啡肽)的表达。结果1内异症患者病灶内的NGF及其受体P75的表达量依次为:宫骶韧带>腹膜>巧囊。TrkA在腹膜中表达最高,其次是宫骶韧带,巧囊表达最少;2宫骶韧带DIE病灶中,中重度痛经组病灶内表达NGF、TrkA及其受体P75较轻度或无痛经症状的宫骶韧带组高,而腹膜及巧囊病灶NGF、TrkA及P75表达量与痛经程度之间无明显相关性;3内异症患者宫骶韧带病灶、腹膜病灶表达NGF、TrkA及P75表达量较非内异症患者高。而巧囊表达NGF及其受体TrkA、P75与非内异症患者比较无显著差异;4重度痛经的患者子宫内膜表达P75较无痛经症状患者及对照组高;5体外培养有痛经症状内异症患者在位内膜基质细胞表达NGF及其受体TrkA较无痛经症状的正常子宫内膜基质细胞高;6体外培养有痛经症状内异症患者在位内膜基质细胞对NGFmRNA及其受体TrkAmRNA、P75mRNA的表达与对照组比较,无显著差异。提示在位内膜基质细胞可能在转录后水平受到影响,从而增加了蛋白的表达;7在位子宫内膜基质细胞及正常内膜基质细胞在不同浓度的雌、孕激素作用后表达NGFmRNA、TrkAmRNA及P75mRNA,结果显示干预前后无明显变化;8同一浓度的17β雌二醇干预内异症患者在位内膜及正常内膜基质细胞,药物作用后24小时以内,NGFmRNA、的表达从8小时至24小时期间开始有上升趋势,而对照组有下调趋势;9不同浓度的GnRHa的作用下,异位子宫内膜细胞表达NGFmRNA、TrkAmRNA、P75mRNA明显下降,有显著性差异。而正常的子宫内膜基质细胞,在GnRHa干预后,NGFmRNA、TrkAmRNA、P75mRNA表达的无明显变化。内异症组与对照组比较,差异有显著性;10子宫内膜异位症痛经患者血浆内孤啡肽含量较高,而无痛经患者较低,对照组则更低,NGF在血清中表达趋势同OFQ,且不随月经周期变而改变;11 GnRHa治疗史患者血清内NGF在用药后明显下降,较痛经患者低,甚至较正常人更低,提示使用GnRHa亦可以抑制外周血中的NGF的表达。GnRHa治疗史患者血清OFQ的表达亦较未用药组低。结论1本课题首次研究了常见的几种内异症病灶表达NGF、TrkA及P75表达。发现宫骶韧带DIE内致痛物质增加,重度疼痛较无或轻度疼痛患高。提示了宫骶韧带DIE患者的疼痛可能与NGF及其受体TrkA及P75的表达有关。腹膜与巧囊病灶内表达NGF、TrkA以及P75与疼痛程度无明显相关性,提示腹膜及巧囊患者的疼痛可能与NGF及其受体的表达无关;2体外培养痛经患者在位内膜基质细胞表达NGF及其受体TrkA增加,提示内异症痛经患者在位内膜异常,进一步丰富了“在位内膜决定论”;3体外培养在位子宫内膜基质细胞及正常内膜基质细胞在不同浓度的雌、孕激素作用24小时后表达NGFmRNA、TrkAmRNA及P75mRNA显示干预前后无明显变化,提示NGF在子宫内膜基质细胞的表达可能通过雌、孕激素以外的其他途径产生;4 GnRHa能明显下调内异症患者在位内膜基质细胞表达NGFmRNA、TrkAmRNA及P75mRNA,这可能与GnRHa治疗内异症疼痛的多重机制相关;5血清中NGF及痛觉敏感相关肽在疼痛患者中有增高趋势,提示疼痛患者外周血内可伴有致痛物质的升高,为内异症疼痛的疗效评价提供了新的靶点;6有GnRHa用药史的患者血清内致痛物质下降,说明GnRHa不仅从下丘脑、垂体、性腺轴水平抑制内异症病灶的生长,而且从外周水平下调了患者体内与疼痛相关的因子表达。更多还原

【Abstract】 Pain is one of the most typical symptoms of endometriosis,which affects the life quality of patients of childbearing age.The mechanism of endometirosis pain is not clear. Research has been focused on sensory nerve sensory nerve fibers AS,sensory fibers C in recent years.There are some reports about neural anatomy mechanisms in endometriosis pain.The role of neurotransmitter systems in pathogenesis of endometriosis is still unknown.There are lots of molecules called "pain related substances",such as nerve growth factor(NGF),interleukins and tumor necrosis factor-α.NGF belongs to neurotrophin family,which promotes the survival of neurons.It has been found that the binding of NGF with its receptor causes self-induced phosphorylation and triggers a variety of biological effects,including the release of 5-HT from mast cells,resulting in hyperalgesia,which plays an important role in the regulation of neuropathic and non-neuropathic pain.Different types of lesions related to different pain.Generally speaking,endometrioma is not associated with pain,deep infiltrating endometriosis always involved with dysmenorrhea,CPP,intercourse pain.Literature reports that innervation of ectopic endometrial lesions may related to estrogen sensitive.However,the research about the relationship between different types of endometriosis and its surgery findings have not yet been fully unified.It needs more clinical research.Objective1 To explore NGF and its receptor TrkA,P75 expression in different types of endometriosis lesions,as well as the relationship between the degree of pain;2 To explore the expression of NGF and its receptor TrkA,P75 in eutopic endometrial. In different groups and hope it can enrich the "eutopic endometrial determinism";3 To investigate the normal endometrial and eutopic endometrial in vitro,to quantitative the expression of NGF,TrkA,and P75NTR.And to observe if the expression of NGF,TrkA,and P75NTR can be change by estrogen and progesterone and GnRHa.Materials and Methods1.Immunohistochemistry and double immunofluorescence assay were performed to observe NGF,TrkA and P75 of endometriosis lesions.2.Western Blot and RT-PCR were performed to detect NGF,TrkA and P75 in endometriosis lesions and eutopic endometrium.3.Real-timePCR were performed to detect NGF,and P75 in endometriosis lesions as well as eutopic endometrium.4.Culture endometrium of patients with endometriosis and the control for detection of NGF,TrkA and P75,RT-PCR,cell immunofluorescence,flow cytometry will be used.5.Different concentrations of estrogen and progesterone and GnRHa will be used to stimulate eutopic endometrial and normal endometrial cells in vitro.To detect NGFmRNA,TrkAmRNA and P75mRNA expression in the cell level.6.Use the same concentration of estrogen,RT-PCR assay NGF,TrkA and P75 expression in the cell level at different time points.7.To detect NGF and pain-related peptide(orphanin FQ) expression in serum of patient by ELISA.Results1.The semi-quantity of NGF and its receptor P75 in endometriosis lesions is as follows: uterosacral ligament > retroperitoneal > endometrioma.TrkA expression in the peritoneum is the highest,followed by uterosacral ligament and endometrioma.2.DIE uterosacral ligament lesions with severe dysmenorrhea expressed NGF,TrkA and its receptor P75 and it it higer than the patient with none-mild dysmenorrheal.3.NGF,TrkA and P75 expression in terosacral ligament lesions in patient with endometriosis,which is higer than non-endometriosis patients.Endometrioma does not express more NGF and its receptor TrkA,P75 of than non-endometriosis patients.4.Expression of P75 in patients’ endometrium with severe dysmenorrhea is higher than none -mild dysmenorrheal group and the control group.5.Eutopic endometrial stromal cells from severe dysmenorrheal patient in vitro express more NGF and its receptor TrkA than none-mild dysmenorrheal and normal group.6.NGFmRNA and its receptor TrkAmRNA,P75mRNA expressed by eutopic endometrial stromal cells from severe dysmenorrheal patient in vitro is the same with none-mild dysmenorrheal and normal group.7.The level of NGFmRNA,TrkAmRNA and P75mRNA expressed by eutopic endometrial stromal cells and normal endometrial stromal cells showed no significant change before and after intervention by different concentrations of estrogen, progesterone.8.After administratimg 17βestradiol in stromal cellsof patients with endometriosis and normal endometrial eutopic endometrial stromal cells,the NGFmRNA,from 8 to 24 hours showed an upward trend,while the control group has a downward trend.9.Different concentrations of GnRHa significantly decreased the level of NGFmRNA, TrkAmRNA,P75mRNA in ectopic endometrial cells.There are significant differences compared to the control and the normal group.10.Dysmenorrhea endometriosis patients have a higher level of nociceptin.NGF expression in the serum has the same trend with OFQ.11.Serum NGF decreased in the post-drug period as well as the serum OFQ expression in patients with a history of GnRHa treatment.Conclusions:1.This is the first report of the expression of NGF,TrkA and P75 in endometriosis lesions,which is related to different part of endometriosis lesions and the severity of pain.2.NGF and its receptor TrkA is expressed more in eutopic endometrial stromal cells, suggesting that endometriosis eutopic endometrial is abnormal in patients with dysmenorrhea,and further enrich the "eutopic endometrial determinism".GnRHa may have multiple mechanisms in the treatment of pain.3.Pain substances may be elevated in peripheral blood of patients with endometriosis who have severe dysmenorrhea.GnRHa administration can decreased substance, suggesting that it can reduce pain-related factor expression through multiple ways.更多还原