本文選題：子宮內(nèi)膜異位癥　切入點(diǎn)：多配體蛋白聚糖-1　出處：《鄭州大學(xué)》2014年碩士論文

【摘要】：背景和目的 子宮內(nèi)膜異位癥(endometriosis, EMS)是指子宮內(nèi)膜腺體和間質(zhì)種植在子宮腔以外引起的一種良性疾病。其發(fā)病機(jī)制至今尚未明了，EMS表現(xiàn)出鄰近器官種植和破壞，周?chē)M織的侵潤(rùn)，遠(yuǎn)處部位轉(zhuǎn)移，無(wú)限制的生長(zhǎng)，治療徹底更困難和快速?gòu)?fù)發(fā)等常見(jiàn)的生物學(xué)行為，是具有類(lèi)似惡性腫瘤生物學(xué)特質(zhì)的一種良性病變，因此影響腫瘤細(xì)胞脫離、遷移、粘附、降解及浸潤(rùn)增殖過(guò)程的相關(guān)各種分子被廣泛應(yīng)用于EMS的研究。 多配體蛋白聚糖-1(Syndecan-1, SDC-1)屬于跨膜黏蛋白聚糖家族成員，，是一個(gè)經(jīng)典的生長(zhǎng)因子、血管生成因子、趨化因子，其硫酸肝素側(cè)鏈(heparansulfate, HS)鏈具有廣泛的結(jié)構(gòu)異質(zhì)性，結(jié)合細(xì)胞外基質(zhì)(extra cellular matrix,ECM)、細(xì)胞表面黏附分子、生長(zhǎng)因子、酶等，參與細(xì)胞增殖、黏附、遷移和血管生成等過(guò)程。人類(lèi)白細(xì)胞抗原G(humanleucocyteantigen-G, HLA-G)是非經(jīng)典性的MHC-Ib類(lèi)抗原分子，通過(guò)抑制NK細(xì)胞和T淋巴細(xì)胞的活性，參與腫瘤細(xì)胞的免疫逃逸。ECM的降解是EMS異位內(nèi)膜細(xì)胞侵襲和種植的主要途徑，而MMPs在ECM的降解中起重要作用�；|(zhì)金屬蛋白酶-9(matrixmetalloproteinase-9, MMP-9)可降解ECM，穿透細(xì)胞的基底膜，通過(guò)與整合素的相互作用，從而加強(qiáng)細(xì)胞-細(xì)胞間的黏附作用，同時(shí)促進(jìn)血管內(nèi)皮細(xì)胞的出芽，利于新生血管的形成。 目前，國(guó)內(nèi)外尚未見(jiàn)報(bào)道關(guān)于聯(lián)合檢測(cè)SDC-1、HLA-G和MMP-9在EMS患者中表達(dá)水平的研究。本實(shí)驗(yàn)通過(guò)免疫組化S-P法(immunohistochemical S-Pmethod)和酶聯(lián)免疫吸附法(enzyme linked immune sorbent assay, ELISA)對(duì)EMS患者異位內(nèi)膜、在位內(nèi)膜組織及血清中SDC-1、HLA-G和MMP-9的表達(dá)水平進(jìn)行研究，分析其表達(dá)水平與EMS臨床分期的關(guān)系，并分析三者之間的關(guān)系，以探討SDC-1、HLA-G和MMP-9在EMS發(fā)病機(jī)制中的作用，為EMS的發(fā)病機(jī)制和臨床研究提供理論依據(jù)。 材料和方法 1研究對(duì)象 1.1隨機(jī)抽取2012年7月至2013年9月我院婦科收治的經(jīng)腹腔鏡及病理診斷證實(shí)為卵巢子宮內(nèi)膜異位囊腫的患者88例。采用1985年美國(guó)生育學(xué)會(huì)修訂的EMS臨床分期標(biāo)準(zhǔn)(R-AFS)進(jìn)行分期：Ⅰ-Ⅱ期44例，Ⅲ-Ⅳ期44例，對(duì)照組為35例同期入院行宮腹腔鏡手術(shù)的子宮縱膈患者。 1.2對(duì)88例卵巢子宮內(nèi)膜異位囊腫標(biāo)本進(jìn)行蘇木素伊紅(HE)染色后篩選納入實(shí)驗(yàn)組60例，Ⅰ-Ⅱ期和Ⅲ-Ⅳ期各30例，取卵巢子宮內(nèi)膜異位囊腫部分囊壁作為標(biāo)本，納入異位內(nèi)膜組；60例EMS患者中48例患者，Ⅰ-Ⅱ期25例，Ⅲ-Ⅳ期23例，因有生育要求同時(shí)行宮腔鏡檢查，排除內(nèi)膜病變，取宮腔在位內(nèi)膜作為標(biāo)本，納入在位內(nèi)膜組；35例子宮縱膈患者的標(biāo)本，排除EMS、子宮內(nèi)膜病變及其它疾病，取子宮正常內(nèi)膜作為標(biāo)本，納入對(duì)照組。 1.3123例均于術(shù)前清晨抽取空腹肘靜脈血。 1.4以上納入標(biāo)準(zhǔn)：(1)育齡期女性，月經(jīng)周期基本正常；(2)3個(gè)月內(nèi)無(wú)激素類(lèi)藥物應(yīng)用史；(3)無(wú)其他系統(tǒng)疾病，尤其排除惡性腫瘤；(4)無(wú)急、慢性炎癥及其他盆腔疾��；(5)所取標(biāo)本時(shí)間均為患者處于月經(jīng)周期的增殖期。 2實(shí)驗(yàn)方法 2.1利用免疫組化S-P法檢測(cè)EMS患者異位內(nèi)膜、在位內(nèi)膜及對(duì)照組中SDC-1、HLA-G和MMP-9的表達(dá)水平。 2.2利用ELISA法檢測(cè)EMS患者及對(duì)照組血清中SDC-1、HLA-G和MMP-9的表達(dá)水平。 3統(tǒng)計(jì)學(xué)方法 所有實(shí)驗(yàn)數(shù)據(jù)均采用SPSS17.0統(tǒng)計(jì)軟件包進(jìn)行統(tǒng)計(jì)分析，定量資料的統(tǒng)計(jì)分析以均數(shù)±標(biāo)準(zhǔn)差(±s)表示；兩組獨(dú)立樣本的比較采用獨(dú)立樣本t檢驗(yàn)；兩兩間比較采用最小顯著性差異法(LSD-t)檢驗(yàn)；多組間比較采用單因素方差(ANOVA)分析；相關(guān)性采用Pearson直線相關(guān)性分析；敏感度和特異度采用應(yīng)用受試者工作特征曲線(ROC)分析。檢驗(yàn)水準(zhǔn)α=0.05。 結(jié)果 1．EMS組和對(duì)照組組織中SDC-1、HLA-G和MMP-9蛋白表達(dá)水平 SDC-1、HLA-G和MMP-9蛋白主要表達(dá)于EMS患者異位內(nèi)膜和在位內(nèi)膜組織的腺上皮細(xì)胞胞質(zhì)中，間質(zhì)細(xì)胞的胞質(zhì)也有少量表達(dá)。 異位內(nèi)膜組、在位內(nèi)膜組和對(duì)照組組織中SDC-1蛋白的平均光密度(OD)值分別為：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值分別為：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值分別為：148.11±22.88、117.57±8.72和83.98±9.74，(F=188.97,P=0.000)。兩兩比較SDC-1、HLA-G和MMP-9蛋白在異位內(nèi)膜組、在位內(nèi)膜組中的平均OD值均高于對(duì)照組(P0.05)，SDC-1、HLA-G和MMP-9蛋白在異位內(nèi)膜組的平均OD值高于在位內(nèi)膜組(P 0.05)。 EMS異位內(nèi)膜組中，SDC-1、HLA-G和MMP-9蛋白在Ⅰ-Ⅱ期的平均OD值分別為：129.33±5.08、139.83±7.38和130.85±15.21，在Ⅲ-Ⅳ期的平均OD值分別為：163.37±12.39、165.55±11.39和165.36±14.76；在EMS在位內(nèi)膜組中， SDC-1、 HLA-G和MMP-9蛋白在Ⅰ-Ⅱ期的平均OD值分別為103.39±8.02、122.64±4.03和110.61±6.67，在Ⅲ-Ⅳ期的平均OD值分別為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異位內(nèi)膜和在位內(nèi)膜組織III-IV期均高于I-II期(P均0.05)。 2．EMS組和對(duì)照組血清中SDC-1、HLA-G和MMP-9的表達(dá)水平 SDC-1、 HLA-G和MMP-9在EMS組血清中的表達(dá)水平分別為：3888.34±607.38μg/L、3888.34±607.38μg/L和15.42±2.61μg/L，在對(duì)照組血清中的表達(dá)水平分別為：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患者血清中的表達(dá)水平高于對(duì)照組(P均0.05)。 EMS組中SDC-1、HLA-G和MMP-9在Ⅰ-Ⅱ期的表達(dá)水平分別為：3463.20±352.24μg/L、9.98±2.30μg/L和14.84±2.871μg/L，在Ⅲ-Ⅳ期的表達(dá)水平分別為：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組血清中III-IV期的表達(dá)水平明顯高于I-II期(P均0.05)。 3．經(jīng)ROC曲線分析表明SDC-1、HLA-G和MMP-9聯(lián)合檢測(cè)診斷EMS的敏感性和特異性分別為93.18%和94.28%，三者聯(lián)合診斷EMS的敏感度和特異度明顯高于單獨(dú)檢測(cè)或者兩兩聯(lián)合檢測(cè)。 4．EMS組和對(duì)照組中SDC-1、HLA-G和MMP-9蛋白之間相關(guān)性分析 EMS異位內(nèi)膜組中SDC-1、 HLA-G與MMP-9的表達(dá)水平均呈正相關(guān)(r=0.338,P=0.008；r=0.302,P=0.019)；SDC-1與HLA-G的表達(dá)水平無(wú)相關(guān)性(r=0.185,P=0.157)。 EMS在位內(nèi)膜組中SDC-1、HLA-G與MMP-9的表達(dá)水平均呈正相關(guān)(r=0.476,P=0.002；r=0.601,P=0.000)；SDC-1與HLA-G的表達(dá)水平無(wú)相關(guān)性(r=0.161,P=0.273)。 對(duì)照組中SDC-1與MMP-9、HLA-G的表達(dá)水平兩兩之間分別無(wú)顯著相關(guān)性(r=0.052,P=0.752；r=0.067,P=0.651；r=0.107,P=0.512)。 EMS組血清中SDC-1、 HLA-G與MMP-9的表達(dá)水平均呈正相關(guān)(r=0.683,P0.05； r=0.456,P0.05)； SDC-1與HLA-G的表達(dá)水平無(wú)相關(guān)性(r=0.212,P＞0.05)。 結(jié)論 1．SDC-1、HLA-G和MMP-9可能參與EMS的發(fā)生、發(fā)展過(guò)程； 2．聯(lián)合檢測(cè)SDC-1、HLA-G和MMP-9對(duì)EMS的無(wú)創(chuàng)診斷有一定的參考價(jià)值； 3．SDC-1、HLA-G與MMP-9在EMS的發(fā)生發(fā)展過(guò)程中可能有協(xié)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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R711.71

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