SDC-1、HLA-G和MMP-9在子宫内膜异位症中的检测及其意义

发布时间：2018-03-28 02:18

本文选题：子宫内膜异位症　切入点：多配体蛋白聚糖-1　出处：《郑州大学》2014年硕士论文

【摘要】：背景和目的子宫内膜异位症(endometriosis, EMS)是指子宫内膜腺体和间质种植在子宫腔以外引起的一种良性疾病。其发病机制至今尚未明了，EMS表现出邻近器官种植和破坏，周围组织的侵润，远处部位转移，无限制的生长，治疗彻底更困难和快速复发等常见的生物学行为，是具有类似恶性肿瘤生物学特质的一种良性病变，因此影响肿瘤细胞脱离、迁移、粘附、降解及浸润增殖过程的相关各种分子被广泛应用于EMS的研究。多配体蛋白聚糖-1(Syndecan-1, SDC-1)属于跨膜黏蛋白聚糖家族成员，，是一个经典的生长因子、血管生成因子、趋化因子，其硫酸肝素侧链(heparansulfate, HS)链具有广泛的结构异质性，结合细胞外基质(extra cellular matrix,ECM)、细胞表面黏附分子、生长因子、酶等，参与细胞增殖、黏附、迁移和血管生成等过程。人类白细胞抗原G(humanleucocyteantigen-G, HLA-G)是非经典性的MHC-Ib类抗原分子，通过抑制NK细胞和T淋巴细胞的活性，参与肿瘤细胞的免疫逃逸。ECM的降解是EMS异位内膜细胞侵袭和种植的主要途径，而MMPs在ECM的降解中起重要作用。基质金属蛋白酶-9(matrixmetalloproteinase-9, MMP-9)可降解ECM，穿透细胞的基底膜，通过与整合素的相互作用，从而加强细胞-细胞间的黏附作用，同时促进血管内皮细胞的出芽，利于新生血管的形成。目前，国内外尚未见报道关于联合检测SDC-1、HLA-G和MMP-9在EMS患者中表达水平的研究。本实验通过免疫组化S-P法(immunohistochemical S-Pmethod)和酶联免疫吸附法(enzyme linked immune sorbent assay, ELISA)对EMS患者异位内膜、在位内膜组织及血清中SDC-1、HLA-G和MMP-9的表达水平进行研究，分析其表达水平与EMS临床分期的关系，并分析三者之间的关系，以探讨SDC-1、HLA-G和MMP-9在EMS发病机制中的作用，为EMS的发病机制和临床研究提供理论依据。材料和方法 1研究对象 1.1随机抽取2012年7月至2013年9月我院妇科收治的经腹腔镜及病理诊断证实为卵巢子宫内膜异位囊肿的患者88例。采用1985年美国生育学会修订的EMS临床分期标准(R-AFS)进行分期：Ⅰ-Ⅱ期44例，Ⅲ-Ⅳ期44例，对照组为35例同期入院行宫腹腔镜手术的子宫纵膈患者。 1.2对88例卵巢子宫内膜异位囊肿标本进行苏木素伊红(HE)染色后筛选纳入实验组60例，Ⅰ-Ⅱ期和Ⅲ-Ⅳ期各30例，取卵巢子宫内膜异位囊肿部分囊壁作为标本，纳入异位内膜组；60例EMS患者中48例患者，Ⅰ-Ⅱ期25例，Ⅲ-Ⅳ期23例，因有生育要求同时行宫腔镜检查，排除内膜病变，取宫腔在位内膜作为标本，纳入在位内膜组；35例子宫纵膈患者的标本，排除EMS、子宫内膜病变及其它疾病，取子宫正常内膜作为标本，纳入对照组。 1.3123例均于术前清晨抽取空腹肘静脉血。 1.4以上纳入标准：(1)育龄期女性，月经周期基本正常；(2)3个月内无激素类药物应用史；(3)无其他系统疾病，尤其排除恶性肿瘤；(4)无急、慢性炎症及其他盆腔疾病；(5)所取标本时间均为患者处于月经周期的增殖期。 2实验方法 2.1利用免疫组化S-P法检测EMS患者异位内膜、在位内膜及对照组中SDC-1、HLA-G和MMP-9的表达水平。 2.2利用ELISA法检测EMS患者及对照组血清中SDC-1、HLA-G和MMP-9的表达水平。 3统计学方法所有实验数据均采用SPSS17.0统计软件包进行统计分析，定量资料的统计分析以均数±标准差(±s)表示；两组独立样本的比较采用独立样本t检验；两两间比较采用最小显著性差异法(LSD-t)检验；多组间比较采用单因素方差(ANOVA)分析；相关性采用Pearson直线相关性分析；敏感度和特异度采用应用受试者工作特征曲线(ROC)分析。检验水准α=0.05。结果 1．EMS组和对照组组织中SDC-1、HLA-G和MMP-9蛋白表达水平 SDC-1、HLA-G和MMP-9蛋白主要表达于EMS患者异位内膜和在位内膜组织的腺上皮细胞胞质中，间质细胞的胞质也有少量表达。异位内膜组、在位内膜组和对照组组织中SDC-1蛋白的平均光密度(OD)值分别为：146.35±19.56、114.86±13.67和70.30±11.23，(F=277.44,P=0.000)；HLA-G的平均OD值分别为：152.69±16.08、129.06±8.93和61.95±9.40，(F=654.84,P=0.000)；MMP-9的平均OD值分别为：148.11±22.88、117.57±8.72和83.98±9.74，(F=188.97,P=0.000)。两两比较SDC-1、HLA-G和MMP-9蛋白在异位内膜组、在位内膜组中的平均OD值均高于对照组(P0.05)，SDC-1、HLA-G和MMP-9蛋白在异位内膜组的平均OD值高于在位内膜组(P 0.05)。 EMS异位内膜组中，SDC-1、HLA-G和MMP-9蛋白在Ⅰ-Ⅱ期的平均OD值分别为：129.33±5.08、139.83±7.38和130.85±15.21，在Ⅲ-Ⅳ期的平均OD值分别为：163.37±12.39、165.55±11.39和165.36±14.76；在EMS在位内膜组中， SDC-1、 HLA-G和MMP-9蛋白在Ⅰ-Ⅱ期的平均OD值分别为103.39±8.02、122.64±4.03和110.61±6.67，在Ⅲ-Ⅳ期的平均OD值分别为126.33±6.57、135.49±7.77和124.54±3.10。SDC-1、HLA-G和MMP-9蛋白的平均光度值在EMS异位内膜和在位内膜组织III-IV期均高于I-II期(P均0.05)。 2．EMS组和对照组血清中SDC-1、HLA-G和MMP-9的表达水平 SDC-1、 HLA-G和MMP-9在EMS组血清中的表达水平分别为：3888.34±607.38μg/L、3888.34±607.38μg/L和15.42±2.61μg/L，在对照组血清中的表达水平分别为：1481.91±482.49μg/L、5.77±1.82μg/L和9.01±3.15μg/L；SDC- 1、HLA-G和MMP-9在EMS患者血清中的表达水平高于对照组(P均0.05)。 EMS组中SDC-1、HLA-G和MMP-9在Ⅰ-Ⅱ期的表达水平分别为：3463.20±352.24μg/L、9.98±2.30μg/L和14.84±2.871μg/L，在Ⅲ-Ⅳ期的表达水平分别为：4313.48±502.44μg/L、11.50±1.54μg/L和16.01±2.20μg/L；SDC-1、HLA-G和MMP-9在EMS组血清中III-IV期的表达水平明显高于I-II期(P均0.05)。 3．经ROC曲线分析表明SDC-1、HLA-G和MMP-9联合检测诊断EMS的敏感性和特异性分别为93.18%和94.28%，三者联合诊断EMS的敏感度和特异度明显高于单独检测或者两两联合检测。 4．EMS组和对照组中SDC-1、HLA-G和MMP-9蛋白之间相关性分析 EMS异位内膜组中SDC-1、 HLA-G与MMP-9的表达水平均呈正相关(r=0.338,P=0.008；r=0.302,P=0.019)；SDC-1与HLA-G的表达水平无相关性(r=0.185,P=0.157)。 EMS在位内膜组中SDC-1、HLA-G与MMP-9的表达水平均呈正相关(r=0.476,P=0.002；r=0.601,P=0.000)；SDC-1与HLA-G的表达水平无相关性(r=0.161,P=0.273)。对照组中SDC-1与MMP-9、HLA-G的表达水平两两之间分别无显著相关性(r=0.052,P=0.752；r=0.067,P=0.651；r=0.107,P=0.512)。 EMS组血清中SDC-1、 HLA-G与MMP-9的表达水平均呈正相关(r=0.683,P0.05； r=0.456,P0.05)； SDC-1与HLA-G的表达水平无相关性(r=0.212,P＞0.05)。结论 1．SDC-1、HLA-G和MMP-9可能参与EMS的发生、发展过程； 2．联合检测SDC-1、HLA-G和MMP-9对EMS的无创诊断有一定的参考价值； 3．SDC-1、HLA-G与MMP-9在EMS的发生发展过程中可能有协同作用。
[Abstract]:Background and purpose
Endometriosis (endometriosis, EMS) refers to endometrial glands and stroma planting a benign disease caused by outside of the uterine cavity. Its pathogenesis is still not clear, EMS showed the adjacent organs of planting and destruction, surrounding tissue invasion, distant metastases, unlimited growth, thorough treatment more difficult fast and recurrent biological behavior such as common, is a benign lesion with similar biological characteristics of malignant tumor, thus influence tumor cell detachment, migration, adhesion, degradation and related molecular infiltration and proliferation process is widely applied in EMS.
Syndecan -1 (Syndecan-1, SDC-1) is a transmembrane mucin glycan family members, is a classical growth factor, angiogenesis factor, chemotactic factor, its heparan sulfate chains (heparansulfate, HS) chain with structural heterogeneity widely, combined with extracellular matrix (extra cellular matrix, ECM) that cell surface adhesion molecules, growth factors, enzymes, involved in cell proliferation, adhesion, migration and angiogenesis. Human leukocyte antigen G (humanleucocyteantigen-G, HLA-G) is a non classical HLA class MHC-Ib molecules, through inhibition of NK cells and T lymphocyte activity, the degradation of.ECM in immune escape of tumor cells is the main way of planting EMS invasion and ectopic endometrial cells, while MMPs plays an important role in the degradation of ECM. Matrix metalloproteinase -9 (matrixmetalloproteinase-9, MMP-9) can degrade ECM cells through the basement membrane, The interaction with integrin enhances the adhesion between cells and cells, and promotes the bud of vascular endothelial cells, which is beneficial to the formation of new blood vessels.
At present, there is no report about the joint detection of SDC-1, the expression level of HLA-G and MMP-9 in patients with EMS. The experiments of S-P immunohistochemical method (immunohistochemical S-Pmethod) and enzyme linked immunosorbent assay (enzyme linked immune sorbent assay, ELISA EMS) on patients with ectopic endometrium, eutopic endometrium and serum SDC-1 and to study the expression of HLA-G and MMP-9, to analyze the relationship between the expression level and clinical stage of EMS, and analyze the relationship between the three, in order to explore the role of HLA-G and SDC-1, MMP-9 in the pathogenesis of EMS, and provide a theoretical basis for the clinical research on the pathogenesis of EMS.
Materials and methods
1 research objects
1.1 randomly selected from July 2012 to September 2013 in our hospital were treated by laparoscopy and pathology confirmed 88 cases of ovarian endometriosis patients. By 1985 the American Fertility Society revised clinical staging of EMS (R-AFS): stage I - II stage III - IV period in 44 cases, 44 cases, 35 cases in the control group hospitalized Palace laparoscopic surgery patients with uterine mediastinum.
1.2 of 88 cases of ovarian endometriosis cyst were performed hematoxylin and eosin (HE) staining after screening in 60 cases in the experimental group, I - II and III - IV of the 30 cases, the ovarian endometriosis cyst wall as part of specimens included ectopic endometrium group; 60 cases of EMS patients in 48 patients I - II, 25 cases of stage III / IV 23 cases, because fertility requirements hysteroscopic examination, exclude endometrial lesions, the uterine eutopic endometrium as specimens of eutopic endometrium were included; 35 cases of uterine mediastinum in patients without EMS, endometrial lesions and other diseases, the normal uterine endometrium as the samples in control group.
All the 1.3123 cases were taken empty abdominal blood in the early morning.
More than 1.4 criteria: (1) women of childbearing age, menstrual cycle is normal; (2) without hormone medication history within 3 months; (3) without other diseases, especially to exclude malignancy; (4) no acute, chronic inflammation and other pelvic diseases; (5) the specimens when among all patients in the proliferative phase of the menstrual cycle.
2 experimental method
2.1 the expression level of SDC-1, HLA-G and MMP-9 in the eutopic endometrium and the control group was detected by immunohistochemical S-P method.
2.2 the expression level of SDC-1, HLA-G and MMP-9 in serum of EMS patients and control group was detected by ELISA method.
3 statistical method
All the experimental data are used SPSS17.0 statistical package for statistical analysis, the statistical analysis of quantitative data to mean + standard deviation (+ s); two groups were compared using independent sample independent sample t test; 22 were compared using least significant difference method (LSD-t) test; multiple groups were analyzed using one-way ANOVA (ANOVA) analysis; Pearson linear correlation analysis was used for correlation; sensitivity and specificity by using receiver operating characteristic curve (ROC) analysis. A =0.05. level test
Result
Expression of SDC-1, HLA-G and MMP-9 protein in group 1.EMS and control group
SDC-1, HLA-G and MMP-9 proteins were mainly expressed in the cytoplasm of ectopic endometrium and eutopic endometrial tissue of EMS patients, and the cytoplasm of interstitial cells was also expressed in small amounts.
Ectopic endometrium group, the average optical density of SDC-1 protein in eutopic endometrium group and control group (OD) = 146.35 + 19.56114.86 + 13.67 and 70.30 + 11.23 (F=277.44, P=0.000); the average OD value of HLA-G was 152.69 + 16.08129.06 + 8.93 and 61.95 + 9.40 (F=, 654.84, P=0.000) the average OD value of MMP-9; respectively: 148.11 + 22.88117.57 + 8.72 and 83.98 + 9.74 (F=188.97, P=0.000). 22 comparison of SDC-1, HLA-G and MMP-9 protein in ectopic endometrium, eutopic endometrium of the average OD value was higher than that of control group (P0.05), SDC-1, the average OD value of HLA-G and MMP-9 protein in ectopic endometrium the higher than that in eutopic endometrium group (P 0.05).
SDC-1 EMS of ectopic endometrium, respectively, HLA-G and MMP-9 protein in the OD stages I - II: 129.33 + 5.08139.83 + 7.38 and 130.85 + 15.21, respectively in the average OD value of III - IV period: 163.37 + 12.39165.55 + 11.39 and 165.36 + 14.76; SDC-1 EMS in eutopic endometrium group, HLA-G. And the average OD value of MMP-9 protein in stage I and II were 103.39 + 8.02122.64 + 4.03 and 110.61 + 6.67, the average OD value of III - IV stage were 126.33 + 6.57135.49 + 7.77 and 124.54 + 3.10.SDC-1, the average luminosity of HLA-G and MMP-9 protein EMS value in eutopic and ectopic endometrium were higher than III-IV group I-II (P 0.05).
Expression level of SDC-1, HLA-G and MMP-9 in serum of 2.EMS and control groups
The expression levels of SDC-1, HLA-G and MMP-9 in serum of EMS group were 3888.34 + 607.38 g/L, 3888.34 + 607.38 g/L and 15.42 + 2.61 g/L, respectively. The expression levels in serum of control group were 1481.91, 482.49, g/L, 5.77, 1.82, and 9.01.
1, the expression level of HLA-G and MMP-9 in the serum of EMS patients was higher than that of the control group (P 0.05).
SDC-1 group EMS, HLA-G and MMP-9 respectively. The expression level in stages I - II: 3463.20 + 9.98 + 352.24 g/L, 2.30 g/L and 14.84 + 2.871 g/L, respectively. The expression level III - IV period: 4313.48 + 502.44 g/L 11.50 + 1.54 and 16.01 + 2.20 u g/L g/L; SDC-1, HLA-G and MMP-9 III-IV expression levels in serum of group EMS in the period was higher than that of stage I-II (P 0.05).
3. ROC curve analysis showed that the sensitivity and specificity of combined detection of SDC-1, HLA-G and MMP-9 for diagnosing EMS were 93.18% and 94.28%, respectively. The sensitivity and specificity of three combined diagnosis of EMS were significantly higher than those of single or 22 combined detection.
Analysis of the correlation between SDC-1, HLA-G and MMP-9 protein in group 4.EMS and control group
In the EMS ectopic endometrium group, SDC-1 and HLA-G were positively correlated with the expression level of MMP-9 (r=0.338, P=0.008, r=0.302, P=0.019), and there was no correlation between SDC-1 and HLA-G expression (r=0.185, P=0.157).
In EMS, there was a positive correlation between SDC-1, HLA-G and MMP-9 expression in r=0.476 group (r=0.476, P=0.002, r=0.601, P=0.000), and there was no correlation between SDC-1 and HLA-G expression (r=0.161, SDC-1).
There was no significant correlation between SDC-1 and MMP-9, HLA-G expression level 22 in the control group (r=0.052, P=0.752; r=0.067, P=0.651; r=0.107, P=0.512).
The expression level of SDC-1 and HLA-G in serum of EMS group was positively correlated with MMP-9 expression (r=0.683, P0.05, r=0.456, P0.05), and there was no correlation between SDC-1 and HLA-G expression level (r=0.212, SDC-1 > 0.05).
conclusion
1.SDC-1, HLA-G and MMP-9 may be involved in the occurrence and development of EMS.
2. the combined detection of SDC-1, HLA-G and MMP-9 has a certain reference value for the non invasive diagnosis of EMS.
3.SDC-1, HLA-G and MMP-9 may have synergistic effects during the development of EMS.

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

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