【作者】 朱军；

【导师】 吴瑞瑾；

【作者基本信息】 浙江大学 ， 妇产科学， 2007， 硕士

【摘要】 研究背景子宫内膜异位症(Endometriosis，EMs)是中青年妇女的常见病、多发病，近年来其发病率有明显上升趋势。它病变广泛、形态多样、极具侵袭性和复发性，是难治之症。它的发病机制众说纷纭，但仍以经血逆流种植学说为主导理论，而“在位内膜决定论”观点也正引起众多学者重视。子宫内膜细胞必须突破腹腔液、巨噬细胞、腹膜细胞外基质“三道防线”，才能完成“粘附-侵袭-血管形成”的“三步曲”。近年来，子宫内膜异位症的免疫机制正日益受到关注。巨噬细胞是一种多功能的免疫细胞，其数量和功能异常可能是子宫内膜异位症发病的重要原因之一。Khaleque等研究检测子宫内膜异位症患者在位和异位子宫内膜以及异位内膜临近腹膜组织中巨噬细胞浸润数量，并与正常子宫内膜作对照，结果显示子宫内膜异位症患者的在位内膜巨噬细胞数量比对照组显著增加，Ⅰ、Ⅱ期患者尤为明显。在位内膜巨噬细胞含量随着月经周期逐渐增加，分泌期显著高于增生期。巨噬细胞具有吞噬、代谢、产生细胞因子及抗肿瘤免疫等多种功能，在宿主防御机制及维持机体内环境平衡方面有重要作用。有研究显示巨噬细胞的数量和功能异常是子宫内膜异位症发病的核心环节。子宫内膜异位症患者腹腔液及在位、异位内膜巨噬细胞含量增多，抗原递呈能力以及吞噬能力减弱，分泌功能增强，活化巨噬细胞分泌的细胞因子又起到了推波助澜的作用，而巨噬细胞的产物对自身又有趋化和激活作用。从而引起“瀑布效应”。巨噬细胞通过分泌多种活性因子作用于多种靶细胞，彼此间相互促进或拮抗，形成复杂的细胞—细胞因子网络，诱导腹腔内炎症的发生，改变腹腔微环境，促进异位内膜细胞增殖、浸润、生长及血管生成，参与子宫内膜异位症的发生发展。白介素是巨噬细胞的重要分泌产物。有研究证实子宫内膜异位症腹腔液中IL-1，-6，-8，-10的浓度明显高于正常对照组，活化的巨噬细胞正是其重要来源。其中IL-1靶细胞种类繁多，在细胞因子网络中发挥多种生物学功能如免疫调节、刺激血管生成、防御作用及急性期炎症反应等。IL一1β蛋白水平在子宫内膜异位症患者的异位内膜、腹腔液中均显著升高。除了其本身引发的免疫炎症反应参与子宫内膜异位症的发生外，IL一1β还可通过诱导其他的细胞因子，如整合素、基质金属蛋白酶3，4、白细胞介素8、肿瘤坏死因子和血管内皮生长因子等，促进异位内膜细胞的黏附、侵袭和新生血管的形成，因而在子宫内膜异位症的发生、发展中起重要作用。IL-8对子宫内膜异位症盆腔内炎症、粘连产生及异位病灶中新生血管的生成进而病灶种植、生长均有重要作用。但迄今为止，国内外文献仅限于子宫内膜异位症患者腹腔液中IL-1β和IL-8表达的研究，尚未见对子宫内膜异位症患者在位、异位内膜组织局部巨噬细胞胞内特异抗体CD68、巨噬细胞分泌产物IL-1β，-8的表达及其相关性的研究。研究目的本课题拟在蛋白水平检测巨噬细胞(CD68)在子宫内膜异位症组织局部表达及其与临床分期的关系，并进一步探讨CD68与巨噬细胞分泌的细胞因子IL-1β、IL-8之间的相关性，旨在明确巨噬细胞在异位子宫内膜局部的表达及其功能改变。材料和方法40例卵巢子宫内膜异位囊肿以及同一患者在位内膜标本来自2003-2006年浙江大学医学院附属妇产科医院及温岭市第一人民医院手术切除的存档蜡块；20例因子宫肌瘤等妇科手术患者在位内膜标本来自同时期温岭市第一人民医院手术切除的存档蜡块进行正常对照。组织均经10％甲醛固定。常规石蜡包埋，厚4um连续切片，分别作HE染色和免疫组织化学染色，采用免疫组织化学二步法，在蛋白水平测定CD68，IL-1β，IL-8在非子宫内膜异位症患者子宫内膜和子宫内膜异位症患者在位及异位内膜中的表达及半定量，以卵巢子宫内膜异位囊壁内膜和在位子宫内膜进行比较，分析其与子宫内膜异位症临床分期的关系，并探讨CD68与IL-1β，IL-8之间的相关性。所有数据均采用SPSS13.0统计软件进行统计学分析。结果(1)CD68表达于子宫内膜间质细胞，子宫内膜异位症患者在位子宫内膜阳性率显著高于对照组子宫内膜(P＜0.05)；子宫内膜异位症患者异位子宫内膜阳性率显著高于在位子宫内膜(P＜0.01)；重度子宫内膜异位症患者异位子宫内膜阳性率显著高于轻度患者(P＜0.01)；(2)IL-1β表达于所有子宫内膜间质和腺体细胞，子宫内膜异位症患者异位子宫内膜表达强于在位子宫内膜(P＜0.001)；子宫内膜异位症患者在位子宫内膜表达强于对照组子宮内膜(P＜0.01)；(3)IL-8表达于所有子宫内膜间质和腺体细胞，子宫内膜异位症患者异位子宫内膜表达强于在位子宫内膜(P＜0.01)；子宫内膜异位症患者在位子宫内膜表达与对照组子宫内膜比较无显著性差异(P＞0.05)；(4)子宫内膜异位症患者的在位和异位子宫内膜中CD68与IL-1β、IL-8的表达均呈正相关性(P＜0.05)，在对照组子宫内膜中的表达无相关性(P=0.607)；(5)子宫内膜异位症患者的在位和异位子宫内膜中IL-1β与IL-8表达呈正相关性(P＜0.01)，在对照组子宫内膜中的表达无相关性(P=0.552)。结论(1)巨噬细胞在非子宫内膜异位症患者子宫内膜、轻度及重度子宫内膜异位症患者在位、异位子宫内膜细胞中均有表达，子宫内膜异位症患者的在位、异位子宫内膜中活化巨噬细胞数量较对照组升高，并随疾病严重程度显著上调，提示子宫内膜局部组织内活化巨噬细胞数量可能与子宫内膜异位症病灶的发生有关，并与疾病期别有关。(2)子宫内膜异位症患者异位内膜组织巨噬细胞释放的细胞因子IL-1β，IL-8表达强度较在位子宫内膜及对照组子宫内膜显著增强，并与组织局部活化巨噬细胞含量成正相关性，提示IL-1β，IL-8参与子宫内膜异位病灶的种植和生长，活化的巨噬细胞通过分泌细胞因子如IL-1β，IL-8等参与子宫内膜异位症病灶的形成和发展。更多还原

【Abstract】 BackgroundEndometriosis is one of the common gynecological diseases, affecting 10% of women in reproductive age. Recent years, the incidence of endometriosis is everlasting increasing. The characteristics of the pathologic changes in the lesions of this disease include abroad anatomical distributions, the diversity appearance, and the high invasiveness in residence tissue. The high recurrence rate of endometriosis cause it become a difficult curative disease. Though there were several theories represent to this disease, but the exact pathogenesis is still unknown. Sampson’s theory of retrograde menstruation is the most widely accepted theory postulated to explain peritoneal endometriosis. But prevalence of retrograde menstruation does not correlate with the prevalence of endometriosis among women in reproductive age. It is clear that there must be other factors that contribute to the pathogenesis of this disease. Eutopic endometrium determination hypothesis appear in more and more literatures. This hypothesis indicates that the eutopic endometrium of women with endometriosis are innately abnormal and contribute to the establishment and maintenance of the disease. If refluxed endometrial cells can grow successfully in abdominal cavity, it must breakthrough three lines of host defensive mechanisms that are peritoneal fluid, macrophages and peritoneal ECM. I t must accomplished adhesion-invasion-angiogenesistrilogy.In recently years, there are emerging data from the different literatures present the immunological mechanisms hypotheses postulated to explain endometriosis. Macrophages are multifunctional cells play key roles in local immune response. Macrophages can execute diverse functional activities, including phagocytosis and degradation of foreign antigens, matrix dissolution and tissue remodelling, and production and secretion of cytokines, chemokines and growth factors. Macrophages are the first line of local host defense when menstrual debris reflux into peritoneal cavity. There are several evidence suggest that the changes of numbers and functions of macrophage both in peritoneal fluid and in eutopic and ectopic endometrium play a pivotal role in the establishment of the endometriotic implants. Khaleque investigated the distribution of macrophage infiltration in eutopic and ectopic endometrium throughout the menstrual cycle. They found that infiltration macrophages appeared to be higher in the endometrium in women with endometriosis than in those without endometriosis, and this was more marked in minor endometriosis. The eutopic endometrium in women with endometriosis appeared to harbor more macrophages in both the proliferative phase and secretory phase compared with control women. Macrophages infiltration in the endometrium increased steadily throughout the cycle of the endometrium . Several published reports suggested that the elevated numbers and activated function of macrophages in eutopic and ectopic endometrium, in peritoneal fluid (PF) of women with endometriosis play a core role in the pathologenesis of endometriosis. The function of antigen presentation and phagocytosis of activated macrophages are reduced and the secrectory functions are imposed. Activated macrophages may release products such as cytokines, prostaglandins (PGs), complement components and hydrolytic enzymes. Activated macrophages with their liberated cytokines or growth factors induce proliferation of cells, involved in inflammation, tissue repair and angiogenesis, sothat play an active role in the initiation, maintenance and progression of endometriosis. Macrophages product then accumulate and activate more macrophages, thus a positive cascade and cells-cytokines network established that facilitates implantation and growth of ectopic endometrial cells.It was proved that IL- 1, IL-2, IL-6, IL-8 and TNFa are all increased in the PF of women with endometriosis, and these cytokines mainly produced by peritoneal macrophages. Among these factors, interleukin 1 is a pivotal macrophage-derived cytokine which plays a key role in primary immune responses. Levels of IL-1β are elevated in the PF of women with endometriosis and IL-1β has been shown to upregulate cytokines and growth factors such as intergrin, MMP3, MMP4, IL-8, TNF and VEGF, which may contribute to vascularisation, adhesion, invasive and establishment of ectopic endometriotic lesions.IL-8 is a potent angiogenic, proinflammatory, growth-promoting cytokine. It stimulates the growth of ectopic, eutopic endometrial cells and DNA synthesis, and play a major role in ectopic endometrial cell implant. Until now, few data showed in the correlation between the IL-1β , IL-8 in PF of women with endometriosis. The expression of CD68( a specific antibody of the activated macrophage) and IL-1β, IL-8 in ectopic and eutopic endometrium of women with emdometriosis and their correlation remain unreported.Objectives (1 ) To detect the distribution and expression of macrophages inendometrium and to determine the correlation between the tissue accumulation of macrophages and the staging of endometriosis; (2) To investigate the correlation between the macrophages and it’s products of IL-1β ,IL-8 and to clarify the changes of expression and function of macrophages in the lesions of endometriosisMaterials and MethodsMaterials In a series of 40 women with endometriosis undergoing operation, biopsies samples were collected from ovarian endometriosis cystoma and corresponding eutopic endometrium. As a control group, eutopic endometrium from 20 women operated on other gynecologic diseases such as myomas were also evaluated.Methods The immunohistochemical techniques with the antibody for the CD68, IL-1β and IL-8 were performed on paraffin wax sections of endometrial tissue and eutopic endometrium from patients with and without endometriosis . Immunostaining outcome was semi-quantified by the HSCORE. Routine HE staining was accomplished in all samples.Results(1) The infiltration of macrophages as shown by CD68-positive brown spots expressed in eutopic, ectopic endometrium. The number of CD68 positive cells are larger in the eutopic endometrium in women with endometriosis than in those without endometriosis(P<0.05). The ectopic endometrium are infiltrated more macrophages than eutopic endometrium in each group of the endometriosis women (p<0.01). Women with severe endometriosis harbored more macrophages than women with minor endometriosis or control group (P<0.01)(2) IL-1β positive stain in all endometrial endothelial cells and stromal cells. The expression of IL-1β were stronger in the ectopic endometrium than eutopic endometrim in endometriosis women (p<0.001). The expression of IL- 1β were stronger in eutopic endometrim in endometriosis women than in endometrium of women without endometriosis (p<0. 01)(3) IL-8 positive staining expressed in endometrial endothelial cell and stroma. In ectopic endometrium, IL-8 HSCORE values were significantly higher than those in normal eutopic endometrium (P<0.01) . There was no significantdifference in the IL-8 expression between the eutopic endometrium of women with and without endometriosis (P>0.05) .(4) There was a significant correlation between macrophages infiltration and immunoreaction of IL-1β or IL-8 in women with endometriosis( P<0.05). There was no correlation between them in control women (P=0.607) .( 5) There was a significant correlation between IL-1 β and IL-8 in women with endometriosis.( P<0. 01).There was no correlation between them in control women(P=0.552)Conclusions(1) Ectopic and the corresponding eutopic endometrium of women with endometriosis harbor more macrophages than the endometrium of women without endometriosis. The severity of endometriosis is associated with abundant recruitment and infiltration of macrophages, indicating that the activated macrophages in eutopic and ectopic endometrium of women with endometriosis contribute to the establishment, maintenance and advancement of this disease.(2) Endometriotic tissue had significantly higher immunostaining of macrophage-derived cytokines such as IL-1β, IL-8 than endometrium from both patients with and without endometriosis. Suggesting IL-1β and IL-8 may participate in an integrated inflammatory cascade that facilitates implantation and growth of ectopic endometrial cells in endometriosis women.(3) The intensity of immunostaining of IL-1β , IL-8 , and the numbers of macrophages infiltrated in endometriotic tissue were positively correlated, suggesting endometrial macrophages are associated with endometriosis and may play a central role in its etiology by releasing IL-1β, IL-8.更多还原